Methods for treatment and prevention of tauopathies and amyloid beta  amyloidosis by modulating crf receptor signaling

ABSTRACT

Methods for treating or preventing tauopathies and/or Aβ amyloidosis by modulating CRF receptor signaling. Accumulation of hyperphosphorlyated tau protein in the CNS may be reduced by administration of CRF-R1 selective antagonists and/or CRF-R2 selective agonists. For example, in some aspects, methods for preventing the onset of Alzheimer&#39;s disease by administration of CRF-R1 selective antagonist are provided.

This application is a continuation of U.S. application Ser. No.12/663,805, filed Jun. 1, 2010, which is a national phase applicationunder 35 U.S.C. §371 of International Application No. PCT/US2008/066848,filed Jun. 13, 2008, which claims priority to U.S. ProvisionalApplication No. 60/943,672 filed on Jun. 13, 2007, the contents of eachof which are specifically incorporated herein by reference in theirentirety without disclaimer.

This invention was made with government support under grant numberDK026741 awarded by the United States National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention generally concerns protein mediated neurologicaldisorders. In particular, the invention provides new methods to delaythe onset and/or progression of tauopathies.

2. Description of Related Art

A wide variety of neurodegenerative disorders involve accumulation ofinsoluble tau protein. These disorders are collectively known astauopathies due to the presence of tau tangles though, their clinicalmanifestation vary widely. One well-known tauopathy, Alzheimer's disease(AD) is defined neuropathologically by the accumulation of beta-amyloidplaques and neurofibrillary tangles (NFT) containing tau protein. NFTsin AD have been found to consist of hyperphosphorylated forms of the tauprotein. Hyperphosphorylated tau exhibits reduced ability to bind andstabilize microtubules and can self-aggregate to form insoluble pairedhelical filaments (PHFs), which comprise NFTs (Gustke et al., 1992;Bramblett et al., 1993; Alonso et al., 1996). The incidence of NFTs ispositively correlated with cognitive deficit and neuronal loss in AD(Arriagada et al., 1992; Gomez-Isla et al., 1997), and the discoverythat mutations in the tau gene underlie autosomal dominant forms offrontotemporal dementia suggests that pathological changes in tau canserve as a principal cause of neurodegeneration and cognitive impairment(Hutton et al., 1998; Poorkaj et al., 1998; Spillantini et al., 1998).In view of this, tau phosphorylation has been studies as a possiblemediator of tauopathies.

Exposure to a range of environmental insults, or stresses, can activatetau kinases and induce tau phosphorylation (tau-P) in the rodent centralnervous system (CNS) (e.g., Korneyev et al., 1995; Papasozomenos, 1996;Korneyev, 1998; Yanagisawa et al., 1999; Planel et al., 2001, 2004;Arendt et al., 2003; Feng et al., 2005). This effect has been reportedconsistently in the hippocampal formation, a key structure in learningand memory, and the initial site of tau pathology in AD (Braak andBraak, 1991). Although acute stress-induced tau-P is reversible, themechanisms that govern this phenomenon are unknown, and it is not clearwhether and how it may be manifest under chronic stress conditions.Addressing these questions may better define the elusive links betweenthe stress axis and AD-related pathogenic processes, as increasedexposure and/or sensitivity to stress in humans and rodent modelsconfers increased risk of dementia and AD neuropathology (Wilson et al.,2003; Jeong et al., 2006).

Warranting consideration in this respect are glucocorticoids, dominantstress hormones whose elevated levels in aging have been linked toincreased neuronal vulnerability in hippocampus (Sapolsky et al., 1985,1986). However, acute stress-induced tau-P is reportedly unaffected inadrenalectomized mice (Korneyev et al., 1995), suggesting thatglucocorticoid secretion may not be pivotally involved. Alternatively,the corticotropin-releasing factor (CRF) signaling system plays anessential role in initiating pituitary-adrenal responses to stress, andhas been implicated as a transmitter/modulator in CNS systems thatmediate complementary autonomic and behavioral adjustments, earningconsideration as a general mediator/integrator of stress adaptations(Chadwick et al., 1993). CRF and related ligands (urocortins 1-3) exerttheir biological effects via two G-protein coupled receptors (CRF-R1,CRF-R2) that are differentially distributed in brain (Van Pett et al.,2000), and exert convergent effects on a range of stress-relatedendpoints (Bale and Vale, 2004). CRF-R ligands can conferneuroprotection, in vitro, by altering amyloid precursor protein (APP)processing and suppressing tau kinases, and reduced central CRFexpression has been documented early in AD progression (Rehman, 2002;Bayatti and Behl, 2005). Furthermore, studies examining the effects ofCRF-R signaling on apoptotic cell death in neurons indicated that CRF-R2agonists had no effect on neuronal death while CRF-R1 agonist has aprotective effect (Pedersen et al., 2002). These results lead many inthe field to contemplate that CRF-R1 agonist might have use astherapeutics in AD (Pedersen et al., 2002; U.S. Publn. 20030186867).However, to date the precise role of CRF-R signaling in the developmentand progression of tauopathies has remained unclear.

SUMMARY OF THE INVENTION

In a first embodiment the instant invention provides a method fortreating, preventing or delaying the onset (or progression) of atauopathy in a subject. Thus, in certain preferred aspects, theinvention provides methods for delaying the onset or progression of atauopathy comprising administering to the subject an effective amount ofa CRF-R1 selective antagonist and/or an effective amount of a CRF-R2selective agonist. In further aspects, the composition may compriseadministering an effective amount of a CRF-R1 selective antagonistand/or an effective amount of a CRF-R2 selective agonist to the subjectseparately and/or in a single formulation. Thus, in certain cases,method of the invention may involve administering an effective amount ofa CRF-R1 antagonist and a CRF-R2 agonist. As used herein the termtauopathy refers to a neurodegenerative disease that involves theformation of pathology involving alterations in tau protein, such asneurofibrillary tangles (NFTs). Thus, in some cases, a tauopathy may beAlzheimer's disease (AD), Amyotrophic lateralsclerosis/parkinsonism-dementia complex, Argyrophilic grain dementia,Corticobasal degeneration, Creutzfeldt-Jakob disease, Dementiapugilistica, Diffuse neurofibrillary tangles with calcificationa, Down'ssyndrome, Frontotemporal dementia with parkinsonism (linked tochromosome 17), Gerstmann-Straussler-Scheinker disease,Hallervorden-Spatz disease, Myotonic dystrophy, Niemann-Pick disease(type C), Non-Guamanian motor neuron disease with neurofibrillarytangles, Pick's disease, Postencephalitic parkinsonism, Prion proteincerebral amyloid angiopathy, Progressive subcortical gliosis,Progressive supranuclear palsy, Subacute sclerosing panencephalitis orTangle only dementia. For instance, in certain aspects the inventionprovides methods and compositions for delaying the onset or progressionof AD. However, in certain other aspects, methods for treating, delayingthe onset or delaying the progression of a non-Alzheimer tauopathy areprovided.

In further embodiments the invention provides a method for treating,blocking or delaying the onset and/or progression of an amyloid betaamyloidosis (Aβ amyloidosis) in a subject comprising administering tothe subject a composition comprising an effective amount of a CRF-R1selective antagonist and/or an effective amount of a CRF-R2 selectiveagonist. Thus, in some aspects, method of the invention may involveadministering an effective amount of a CRF-R1 antagonist and a CRF-R2agonist separately and/or in a single formulation. In further aspects,Aβ amyloidosis is a neurodegenerative disease that involves theformation of amyloid beta (Aβ plaques. In still further aspects, the Aβamyloidosis involves production of Aβ by truncation or cleavageprocessing of the amyloid precursor protein (APP). Thus, in someaspects, an Aβ amyloidosis may be AD, Cerebral amyloid angiopathy,Inclusion body myositis, or variants of Lewy body dementia. Forinstance, in certain aspects the invention provides methods andcompositions for delaying the onset or progression of AD. However, incertain other aspects, methods for treating, delaying the onset ordelaying the progression of a non-Alzheimer Aβ amyloidosis are provided.

In some embodiments the invention concerns administration of acomposition (e.g., a CRF-R1 antagonist) to a subject. A subject mayrefer to any animal that is a recipient of the methods or compositionsherein, though in a preferred embodiment, the subject is a human. Theskilled artisan will recognize that in certain cases, the methods of theinvention involve delaying or preventing the progression of a tauopathyand/or an Aβ amyloidosis in a subject. Thus, in certain aspects, asubject may be a subject that has been diagnosed with a tauopathy and/oran AP amyloidosis. For example, a subject may be defined as a person whohas been diagnosed with AD or has clinical and/or pathological signs ofAD. In still other embodiments, there is provided a method forpreventing or delaying the onset of a tauopathy and/or an Aβamyloidosis. Thus, the skilled artisan will recognize in some casessubjects are defined as not having a tauopathy and/or an Aβ amyloidosis.For example, in some aspects, a subject maybe at risk for developing atauopathy and/or an Aβ amyloidosis. An at risk subject may for instance,have a genetic predisposition to a tauopathy and/or an Aβ amyloidosis(e.g., as ascertained by family history or a genetic mutation). In stillfurther cases, an at risk subject may lack clinical disease but compriserisk factors for disease such as declining cognitive (e.g., mildcognitive impairment (MCI)) or memory function or elevated levels of amarker protein (e.g., tau or amyloid beta) in the serum or CNS orincreased or advancing age.

Thus, in some preferred embodiments, there is provided a method fordelaying the onset or progression of a tauopathy and/or an Aβamyloidosis in a subject comprising administering an effective amount ofa CRF-R1 selective antagonist. As used herein the phrase CRF-R1selective antagonist means an antagonist that is more effective atantagonizing CRF-R1 signaling than CRF-R2. For example, in certaincases, a CRF-R1 selective antagonist has between about 10 and about 100,1000, or 10,000 fold more antagonist activity on CRF-R1 than on CRF-R2.For example, a selective antagonist may be defined as molecules thatbinds to CRF-R1 with a 10, 100, 1000 or 10,000 fold higher affinity thanit binds to CRF-R2. Thus, in some cases, a CRF-R1 selective antagonisthas essentially no CRF-R2 antagonist activity as exemplified byantalarmin. In still other cases a CRF-R1 selective antagonist maycomprise CRF-R2 agonist activity. Furthermore, a CRF-R1 antagonist asdescribed herein may also comprise molecules that reduced expression ofCRF-R1 thereby antagonizing the CRF-R1 signaling pathway. For example,in certain aspects a CRF-R1 antagonist may be a CRF-R1 selective siRNA.

In still further embodiments there is provided a method for delaying theonset or progression of a tauopathy and/or an Aβ amyloidosis in asubject comprising administering an effective amount of a CRF-R2selective agonist. As used herein the phrase CRF-R2 selective agonistrefers to an agonist that is more effective at agonizing CRF-R2 thanCRF-R1. For example, in certain cases, a CRF-R2 selective agonist hasbetween about 10 and about 100, 1000, or 10,000 fold more agonistactivity on CRF-R2 than CRF-R1. For example, a selective agonist may bedefined a molecule that binds to CRF-R2 with about a 10, 100, 1000 or10,000 fold higher affinity than it binds to CRF-R1. Thus, in somecases, a CRF-R2 selective agonist has essentially no CRF-R1 agonistactivity. In still other cases, a CRF-R2 selective agonist comprisesCRF-R1 antagonist activity. Furthermore, a CRF-R2 agonist as describedherein may also comprise an indirect agonist such as molecules thatincreases expression of CRF-R2 thereby agonizing the CRF-R2 signalingpathway.

As described here, in certain aspects the invention concernsadministering an effective amount of a CRF-R1 selective antagonistand/or a CRF-R2 selective agonist. The skilled artisan will readilyunderstand that an important aspect of the invention is the selectivityof a particular agonist or antagonist. Thus, depending on the level ofselectivity of a particular CRF-R agonist or antagonist the effectiveconcentration for administration may vary. Furthermore, as describedsupra, an important marker for the effectiveness of a CRF-R agonist orantagonist is reduction of stress-induced tau phosphorylation orreduction of stress-induced insoluble tau protein accumulation or theformation of Aβ plaques. Nonetheless, transient tau phosphorylation maybe an important mediator of stress response and thus complete abrogationof phosphorylation is not preferred. Hence, in some aspects, aneffective dosage is defined a dosage that partially reducesstress-induced tau protein phosphorylation such as dosage that reducestau phosphorylation by between about 90%, 80%, 70%, 60%, 50%, 40%, 30%or 20% and 10% following stress. In other aspects, an effective dosageis defined a dosage that reduce amyloidosis by between about 90%, 80%,70%, 60%, 50%, 40%, 30% or 20% and 10%. Thus, in certain cases, theinstant invention provides a method for determining an effective amountof a CRF-R1 antagonist or a CRF-R2 agonist dosage by assessing areduction of stress-induced tau phosphorylation and/or a reduction in Aβamyloidosis. For example, a CRF-R1 antagonist with essentially no CRF-R2agonist activity such as antalarmin may be administered in a dosageequivalent to a murine dosage of between about 2 mg/kg and 200 mg/kg(e.g., about 6 to about 600 mg/m²), such as a dose of about 20 mg/kg(e.g., about 60 mg/m²).

Methods of administering a CRF-R1 antagonist or CRF-R2 agonist of theinvention will depend upon the particular type of agonist or antagonistused. In general, a CRF-R1 selective antagonist or a CRF-R2 selectiveagonist may be administered by any method known in the art such astopically, intravenously, intradermally, intraarterially,intraperitoneally, intracranially, intrathecally,intracerebroventricularly, mucosally, intraocularally, subcutaneously ororally. In certain aspects, molecules or compositions with the abilityto cross the blood-brain barrier (BBB) may be administered topically,intravenously, intradermally, intraarterially, intraperitoneally,mucosally, intraocularally, subcutaneously or orally. On the other hand,in some cases, molecules or compositions unable to traverse the BBBmaybe administered directly to the CNS. In some very specific aspects,the molecules or compositions directly administered to the CNS can beadministered intrathecally, intracerebroventricularly, orintracranially.

In some very specific aspects of the invention a CRF-R1 specificantagonist may comprise a peptide or polypeptide antagonist, such as amodified CRF ligand polypeptide. Furthermore, in certain aspects, aCRF-R1 selective antagonist may be a CRF-R1 binding antibody or afragment thereof. In still other aspects of the invention a CRF-R1selective antagonist comprises a small molecule antagonist. For example,a CRF-R1 antagonist may be TIMP, DMP696, DMP904, CRA 1000, CRA 1001,SSR125543A, SN003, DMP695, NBI 27914, NBI 30775, NBI 34041, NBI 35965,CP154,526, R121919, R121920, LWH234, antalarmin or a derivative thereof.In a highly preferred embodiment the CRF-R1 antagonist is awater-soluble antagonist that may be administered orally.

In still further specific aspects of the invention a CRF-R2 specificagonist for use according to the invention comprises a peptide orpolypeptide CRF-R2 agonist. For example, a CRF-R2 peptide agonist may bea Ucn 2 or Ucn 3 peptide or a derivative thereof. A number of Ucn 2 andUcn 3 derived peptides comprising selective CRF-R2 agonist activity havebeen developed and may be used according to the invention see forexample U.S. Pat. Nos. 6,953,838 and 6,838,274 and Mazur et al. (2005).Furthermore, in certain aspects a CRF-R2 selective agonist may be aCRF-R2 binding antibody or a fragment thereof. In still further aspectsof the invention, a CRF-R2 selective agonist for use according to theinvention is a small molecule agonist.

In yet further embodiments of the invention a CRF-R1 selectiveantagonist and/or a CRF-R2 selective agonist may further comprises a CNStargeting agent. As used herein CNS targeting merely refers to an agentthat increases the amount of a composition or molecule in the CNS. Forexample, in some aspects, a CNS targeting agent is a polypeptide such asan antibody or cationized albumin. Thus, polypeptide CNS targetingagents may in some aspects, be bound to a CRF-R agonist or antagonistfor use according to the invention. In some very specific cases, apeptide (or polypeptide) CRF-R agonist or antagonist may be provided asa fusion protein with a CNS targeting polypeptide. For example, a CNStargeting polypeptide may be an antibody that mediates transcytosisacross the BBB. Such an antibody may comprise, for example, a monoclonalantibody to transferrin receptor (e.g., OX26) or monoclonal antibodiesto the insulin receptor (Schnyder & Huwyler, 2005). In certain othercases nanoparticles such as Polysorbate 80-coated polybutylcyanoacrylatenanoparticles may be used to deliver compositions to the CNS (Olivier,2005). In still further aspects, CNS targeting polypeptides may beconjugated to liposomes to form CNS targeting complexes (Schnyder &Huwyler, 2005). Furthermore, peptide and polypeptide CRF-R1 antagonistsand/or CRF-R2 agonists may be targeted to the CNS by glycosylation, forexample as described in Egleton & Davis (2005).

In still further embodiments of the invention there is provided a methodfor identifying an agent for treating, preventing the onset orpreventing progression of a tauopathy comprising: (a) administering acandidate agent to an animal; (b) subjecting the animal to a stress; (d)determining tau phosphorylation, dephosporylation (e.g., PP2A-c, othertau phosphatase protein level, or tau kinase activity) or insoluble tauaccumulation in the CNS following stress wherein a decreased in tauphosphorylation, a decrease in PP2A-c level or decreased insoluble tauaccumulation in animals treated with a candidate agent relative tocontrol animals is indicative of activity in treating, preventing theonset or preventing progression of a tauopathy. In certain aspects,methods of the invention may involve determining tau phosphorylation,PP2A-c level or insoluble tau accumulation in a hippocampal tissuefollowing stress. Furthermore, in certain aspects, determining adecrease tau phosphorylation or insoluble tau protein may comprisemeasuring the amount of tau phosphorylation or a level of insoluble tauprotein accumulation. In still further aspects a method may furthercomprise between steps (b) and (d), step (c): repeating steps (a) and(b) to determine the effect of a candidate agent during repeated/chronicstress. Thus, in some aspects steps (a) and (b) may be repeated 2, 3, 4,5, 6, 7, 8, 9, 10, 20, 50, 100 times or more. In further aspects steps(a) and (b) comprise exposing animals to a series of chronic variablestress. The skilled artisan will recognize that in some aspects such amethod may be used to determine an effective amount of a CRF-R1antagonist or CRF-R2 agonist to reduce tau phosphorylation by betweenabout 90%, 80%, 70%, 60%, 50%, 40%, 30% or 20% and 10% following stress.

In certain preferred aspects an animal for use according to theinvention is a rat or a mouse. Various stress conditions maybe used inmethods of the invention. For example, a stress may be a physical stressor, more preferably, an emotional stress. In certain preferred aspects arelatively mild stress, such as physical restraint stress is used. Instill further cases, heat shock, forced swimming, starvation or exposureto anesthetics may be used to apply stress to animals.

Methods for determining tau phosphorylation, or tau phosphatase (PP2A-c)protein level or insoluble tau accumulation are well known in the art.For example, tau phosphorylation is by determined by biochemical,anatomical or antigen capture approaches such as binding of aphosphorylation specific antibody (e.g., in a Western blot,immunohistochemistry or ELISA). Likewise, in certain aspects, antibodybinding may be used to determine PP2A-c protein level, for example afterrepeated stress. Methods for determining insoluble tau proteinaccumulation are also well known in the art. For example, insoluble tauprotein may be determined by detergent extraction and detection byantibody binding. Furthermore, insoluble tau accumulation may bedetermined by detecting tau aggregates, filaments or tangles for exampleby microscopy (e.g., electron microscopy) or by scanning techniques suchas Positron Emission Tomography (PET) scan or Magnetic Resonance Imaging(MRI).

Methods for determining Aβ plaques or Aβ peptide level are well known inthe art. For example, Aβ plaques are detected by biochemical, anatomicalor antigen capture approaches such as binding of a Aβ specific antibody(e.g., in a Western blot or ELISA). Likewise, in certain aspects,antibody binding may be used to determine Aβ peptide level, for exampleafter repeated stress, chronic variable stress, or after CRF-R1antagonist treatment.

Embodiments discussed in the context of a methods and/or composition ofthe invention may be employed with respect to any other method orcomposition described herein. Thus, an embodiment pertaining to onemethod or composition may be applied to other methods and compositionsof the invention as well.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to thedrawings in combination with the detailed description of specificembodiments presented herein.

FIG. 1: Time course of stress-induced tau phosphorylation. Western blotsof hippocampal extracts probed for tau-P at select AD-relevant sites inunstressed controls (C) and mice sacrificed 0, 20, 40, 60 or 90 minafter a single 30 min exposure to restraint stress. For all epitopesexamined, signal under basal conditions was low, but rose immediatelyafter stress to levels that were markedly increased (2-10 fold) overcontrol levels through 60 min, and diminished thereafter. The 20 minpost-stress time point was adopted for use in subsequent experiments.β-actin was used as a loading control.

FIG. 2A-B: The role of glucocorticoids in stress-induced tau-P. FIG. 2A,Western analysis of hippocampal tau-P at the AT8 and PHF-1 sites undercontrol (C) and acute stress (S) conditions in intact andadrenalectomized (ADX) mice. ADX mice were supplemented withcorticosterone to approximate basal circulating steroid levels. FIG. 2B,Quantitative analysis, expressed as mean±SEM percentage of integratedintensity values of intact unstressed controls, reveals that intact andADX animals manifest comparably robust stress-induced increments intau-P at each site, whose magnitude did not differ significantly. Lightbars and dark bars indicate unstressed and stressed animalsrespectively. **, indicates that results differ significantly fromintact, unstressed controls, P<0.001; ns, non-significant (P>0.05). n=3mice/condition. β-actin was used as a loading control.

FIG. 3A-B: Differential regulation of stress-induced tau-P by CRF-Rstatus. FIG. 3A, phosphorylation responses of seven AD-relevant tausites in hippocampal extracts from wild type (wt), CRF-R1−/− andCRF-R2−/− mice killed at 20 min after acute restraint stress (S) or notreatment (C). FIG. 3B, quantitative analysis revealed that wt animalsdisplayed the expected increases in tau-P following stress at allepitopes. CRF-R1−/− animals displayed elevated basal levels ofphosphorylation at the AT8 and PHF-1 sites, but did not manifestsignificantly increased responses following stress at any site.Conversely, CRF-R2−/− mice showed normal basal levels of tau-P at allsites, but exaggerated responses to stress at five of the seven epitopesassayed. Light bars and dark bars indicate unstressed and stressedanimals respectively. Data are presented as mean±SEM percentage of wtcontrol values. *, differs significantly from unstressed wt condition,P<0.01; **, P<0.001; †, differs from wt stressed group, P<0.05. n=3mice/condition. β-actin was used as a loading control.

FIG. 4A-B: Cellular localization of stress- and peptide-induced tau-P.FIG. 4A, immunoperoxidase staining for PHF-1-immunoreactivity in thedentate gyrus (top) and CA1 field (bottom) of mouse hippocampus as afunction of stress status and genotype. Phosphorylation is localized todistinct perikaryal/dendritic and axonal elements, and, importantly,varies with stress exposure and CRF-R status in a manner identical tothat seen by Western analysis (FIG. 3). FIG. 4B, PHF-1 in the dentategyrus of mice injected icy with synthetic CRF. Tau-P varied as afunction of genotype in a similar fashion to that seen in stressedanimals (FIG. 4A).

FIG. 5A-B: CRF-R1 antagonist blocks stress-induced tau-P. Westernanalysis (FIG. 5A) and quantitation (FIG. 5B) of tau-P at the AT8 andPHF-1 sites in hippocampal extracts from mice pretreated with vehicle orthe small-molecule CRF-R1 antagonist, antalarmin (20 mg/kg, ip.), andsubjected to acute stress or no further treatment, expressed as apercentage of levels in untreated controls (C). At each site, antagonisttreatment did not affect basal levels of phosphorylation, but blockedthe stress-induced increment. The vehicle used for drug administrationhad no significant effect under basal or stress conditions. Light barsand dark bars indicate unstressed and stressed animals respectively. **,differs significantly from unstressed controls, P<0.001; †, differs fromuntreated stressed group, P<0.05. n=3 per condition. β-actin was used asa loading control.

FIG. 6A-B: Modulation of tau kinase activity by stress and CRF-R status.Western analyses using phosphoepitope-specific antibodies to interrogatestress- and/or genotype-dependent variations in the activation state ofglycogen synthase kinase-3 (GSK-3), c-jun N-terminal kinases (JNK46/54)and the extracellular signal-regulated kinases 1 and 2 (ERK1/2);relative levels of cyclin-dependent kinase 5 (cdk5) and its activatorprotein, p35, were also evaluated. The behavior of each kinase grouprecapitulates aspects of the general pattern of tau-P responses in thesame design. Thus, the activated (pY²¹⁶) form of GSK-3β is upregulatedby stress in wt mice, and this response is blocked CRF-R1-deficientanimals and exaggerated in CRF-R2 mutants. The inhibitory (pS⁹) form andtotal (unphosphorylated) GSK3 are unresponsive over these conditions.Both phospho-JNK isoforms were also stress-responsive, and more so inCRF-R2 knockouts; these kinases were distinguished by very high basalphosphorylation levels in CRF-R1−/− mice, which may relate to elevatedtau-P observed in this condition at the AT8 site (see FIG. 3). Levels ofphospho-ERKs, particularly ERK1, were relatively unresponsive. Levels ofcdk5 were stable across the conditions in force here, but its p35activator protein was strongly upregulated by stress in wt and CRF-R2−/−mice. Light bars and dark bars indicate unstressed and stressed animalsrespectively. Data are presented as mean SEM percent of unstressed wtvalues. *, differs significantly from wt control, P<0.01; **, P<0.001.†, differs significantly from wt stress condition, P<0.05. β-actin wasused as a loading control.

FIG. 7A-C: FIG. 7A-B, repeated stress results in chronic elevations andreduced solubility of phosphorylated tau. Western analysis ofhippocampal tau-P at the PHF-1 and AT8 sites of mice sacrificed 20 minor 24 hr after 30 min acute restraint or 14 consecutive daily exposures.Analysis was carried out on both soluble and detergent-solublefractions. Under acute stress conditions tau-P is transient andcontained wholly within the soluble fraction, whereas repeated stressresults in elevated levels of tau-P at both time points, and theappearance of a significant portion of phospho-tau in thedetergent-soluble fraction. These data suggest that the effects ofrepeated emotional stress on tau-P are cumulative, and associated withincreased sequestration in the cellular fraction known to contain thebulk of PHFs in the AD brain. Note that for this analysis, data areexpressed as mean±SEM optical density, rather than as a percentage ofcontrol values, to reflect that lack of detectable signal indetergent-soluble extracts under control conditions. *, differssignificantly from control, P<0.01; **, P<0.001. β-actin was used as aloading control. FIG. 7C, acute and repeated stress effects on PP2A-clevels. Western analysis of relative levels of PP2A-c in mice sacrificed20 min or 24 hr after 30 min acute restraint or 14 consecutive dailyexposures. Under control and acute stress conditions, low levels of freePP2A-c subunit (˜37 kDa) were found within soluble fractions (lanes1-3). Under conditions of repeated stress, PP2A-c protein was elevatedat both time points (lanes 4-5). No PP2A-c signal was observed indetergent-soluble fractions. These data implicate alterations in tauphosphatase activity as contributing to tau-P under repeated stressconditions, at least (cf FIG. 7). Data are expressed as mean±SEM opticaldensity. *, differs significantly from control, P<0.01; **, P<0.001. †,differs significantly from acute 20 min stress condition, P<0.01;††P<0.001. β-actin was used as a loading control.

FIG. 8: CRF-R involvement in stress-induced tau phosphorylation.Schematic summary of the progression of events leading toneurofibrillary tangle formation in AD, adapted from (Drewes, 2004).Indicated upon this are the present findings supporting a differentialinvolvement of CRF-Rs, acting via specific tau kinases, in mediatingacute stress-induced tau-P (circles). While acute stress effects aretransient and readily reversible, repeated stress exposure producescumulative increases in phosphorylated tau, a portion of which issequestered in detergent-soluble fractions. Long-term increases instress exposure and/or sensitivity may result in development of pairedhelical filaments and tangles that represent a defining feature of ADneuropathology.

FIG. 9A-B: Time course and genotype dependence of acute immune challengeeffects on hippocampal tau phosphorylation. FIG. 9A, Western blotsshowing relative levels of tau-P at the PHF-1 site in unstressed mice(NS) and at varying intervals after LPS injection (10 μg/kg, ip). Arobust increment is seen, peaking at 60-90 min after injection. FIG. 9B,Western blots from individual WT and single or double CRFR knockoutmice. Tau-P responses at the PHF-1 site are relatively consistent acrossgenotypes, while those of CRFR2 and double mutants at the AT8 site arevariable, and, on average, suppressed. This pattern of results contrastssharply with those obtained in a restraint stress model, and suggeststhat that distinct circuitry and mechanisms underlie tau-P responses toemotional (restraint) and physiological (LPS) stressors.

FIG. 10: Hippocampal CRFR mRNA and transgenic eGFP expression. Top:CRFR1 (left) and CRFR2 (right) in hippocampus by in situ hybridization.CRFR1 transcripts are expressed in Ammon's horn (CA1, CA3) and the hilarregion of the dentate gyrus, while CRFR2 is weakly expressed throughoutthe principal cell layers. Bottom: Immuno-peroxidase staining for eGFPin a BAC transgenic mouse expressing eGFP under control of the CRFR1promoter (left). The cellular distribution of labeling in hippocampusand cortex is similar to that of CRFR1 mRNA. Axonal projections of CRFR1neurons (bands of punctate labeling) are also prominently anddifferentially labeled in the transgenic mouse. Color images at theright are of neurons in the dentate hilus of an acutely restrainedCRFR1-eGFP mouse co-stained for eGFP and PHF-1 (merged channel at farright). Substantial overlap localizes stress-induced tau-P toCRFR1-expressing hilar neurons.

FIG. 11: Immunoelectron microscopy of tau filaments and aggregates.Left: Paired-helical filament from an AD brain (Braak stage VI)negatively stained and immunogold labeled (black dots) for phospho-tauusing the PHF-1 antibody. Right: Image of (non-immunolabeled) RIPAextract from a mouse subject to 14 d repeated restraint stress (top)shows negatively stained round/globular aggregates (40-100 nm diameter)that were never seen in unstressed, acutely stressed, or solubleextracts of repeatedly stressed mice. These, too, can be decorated byPHF-1-immunogold labeling (bottom), confirming the presence ofphospho-tau. Scale bars=100 nm.

FIG. 12A-B: CRFR-dependence and solubility of repeated stress-inducedtau-P. Western blots detects tau-P at the PHF-1 site in wt, CRFR1 (R1),CRFR2 (R2) and double CRFR (DBL) knockout transgenic mice, which weresacrificed 20 min (20) or 24 hr (24) after acute (A) or repeated (R)restraint stress. Data from unstressed controls (NS) are shown forcomparison. FIG. 12A, Genotype effects. Results in wt mice confirm thatrepeated stress results in persistent accumulations of phospho-tau(compare A24 and R24 lanes). All stress effects seen in wt mice areabolished in R1 and DBL knockouts, and tend to be exaggerated in R2mutants. FIG. 12B, Solubility of phospho-tau as a function of stress andgenotype. Hippocampal extracts from repeatedly stressed mice of eachgenotype and stress condition, separated into soluble (S) and insoluble(INS) fractions. Repeated, but not acute, stress results inaccumulations of phospho-tau in insoluble cellular fractions. Thiseffect is seen in wt and R2-deficient mice, where it tends to beaccentuated (wt vs. R2 group comparison at R24/INS differssignificantly, P<0.01), but not in R1 or DBL knockouts.

FIG. 13: Modulation of Amyloid beta (Aβ) by repeated stress. The bargraphs show the mean levels of AP42 (ELISA) and the Aβ-degrading enzyme,neprilysin (Western analysis), in RIPA hippocampal extracts of micesacrificed at 20 min (20) or 24 hr (24) after their only (acute stress;A) or final of 14 consecutive daily (repeated stress; R) exposure(s) torestraint stress. Acute stress did not affect either measure. Subtle butsignificant reductions in levels of Aβ42, and increases in neprilysinlevels, are seen 24 hr after the last exposure in repeated stress,relative to unstressed controls. *, P<0.05.

FIG. 14: Stress and CRFR1 antagonist effects in a mouse AD model. Groupsof PS/APP mice were subjected to two 14-day exposures to chronicvariable stress at beginning of the fourth and fifth months of age inthe presence or absence of concurrent treatment with the CRFR1antagonist, antalarmin (40 mg/kg/d; ip) or vehicle. Parallel groupswithout exposure to stress were also administered with antalarmin (40mg/kg/d; ip) or vehicle as control groups. All animals were sacrificedat the end of the fifth month of life, shortly after the time at whichamyloid plaques begin to appear in this transgenic mouse line.Comparable fields of the cerebral cortex of the PS/APP transgenic mousemodel of AD were prepared and stained with an antiserum directed againstthe N-terminus of human APP. The density of plaques (arrows) at thistime point was significantly reduced by antagonist treatment in bothstressed and unstressed mice, while stress alone had no significanteffect. Thus, treatment with the CRFR1 antagonist blocked or delayed thedevelopment of amyloid pathology in this mouse AD model.

DETAILED DESCRIPTION OF THE INVENTION

Tauopathies are characterized by CNS accumulation of Tau proteinaggregates know as tangles. Certain tauopathies also involve otherprotein aggregations, such as the CNS plaques comprised of amyloid betaprotein that characterize Alzheimer's disease. Of all tauopathiesAlzheimer's disease has received the greatest amount of attention by themedical research community. For example, many therapies have focused onreducing the neurological symptoms of Alzheimer's disease such as memoryloss. Still other therapies, such as “Alzheimer's vaccines,” that directimmune cells to degrade protein aggregates have also been proposed andare currently under evaluation. Finally, recent studies have suggestedthat therapeutics may be used to prevent toxicity of protein aggregatesby preventing apoptosis in neurons. However, there remains a need forapproaches for reducing accumulation or even initial aggregation tauprotein to prevent clinical manifestation of tauopathies delay theclinical progression of such diseases.

Studies herein extend the range of insults known to provoke tauphosphorylation to include a representative emotional stressor, andprovide evidence bearing on its mechanism (FIG. 8). The abrogation ofstress-induced tau-P in CRF-R1-deficient animals, and the enhancementobserved in CRF-R2 mutants, were paralleled by altered activities ofspecific tau kinases. Furthermore, the demonstration that repeatedstress exerts cumulative effects on tau-P, and results in sequestrationof phospho-tau in insoluble forms suggests a possible mechanism for tauaggregate accumulation in tauopathies. Importantly, the role of CRF-Rsignaling in stress induced tau phosphorylation elucidated hereindicated that CRF-R1 antagonists and CRF-R2 agonists may be used toreduce stress induced tau phosphorylation and resultant insolubleaccumulation of tau. In support of this it was shown that a CRF-R1selective antagonist can reduce stress induced tau phosphorylation inthe brains of test animals (FIG. 5A-B). Thus, the new studies hereinprovide the basis of intervention in the development and progression oftauopathies by using modulators of CRF-R signaling to preventphosphorylated tau protein accumulation. However, the increase in tauphosphorylation induced by physiological stress (e.g. LPS induce immunechallenge) did not appear to be CRFR dependent (FIG. 9A-B). It suggeststhat CRFR involvement may be limited to emotional stressors.

Previously, there has been a lack of therapies that are effective toreduce initial protein aggregation or prevent further accumulation ofaggregated proteins in tauopathies. Therapies that would reduce tauprotein accumulation are of great interest since they have the potentialto delay the onset of disease and/or slow disease progression.Furthermore, therapies that target tau protein accumulation may beapplicable to a wide range of disorder (tauopathies) that involve tauprotein accumulation despite having disparate etiologies, symptoms andsites of action. To this end the instant invention provides methods forantagonizing the CRF-R1 signaling pathway and/or agonizing CRF-R2signaling to reduce stress-induced tau protein phosphorylation. Sincechronic stress is demonstrated to result in accumulation of insolublepermanently phosphorylated tau proteins, methods of the invention may beused to prevent the accumulation or tau protein aggregates and/or toslow the growth of such aggregates. Thus, methods provided herein may beemployed to delay the progression or onset of tauopathy by decreasingthe rate of tau aggregate accumulation.

Previous, in vitro studies regarding the role of CRF-R signaling in ADhad suggested that CRF-R1 agonists might be used to protect neuronalcells from toxic effects of protein aggregates. However, these in vitrostudies failed to recognize a role of CRF signaling in stress inducestau phosphorylation and ultimately in tau accumulation. Thus, theinstant invention also provides an animal model system for screening newmolecules that may be used to modulate stress induced tau proteinphosphorylation. This new system may be used to determine the effect ofcandidate molecules on both transient and chronic stress induced tauprotein phosphorylation and thus provides a platform to identifymolecules can be used to reduce the accumulation of tau protein that mayresult from long term/chronic stress conditions. Furthermore, methodsfor determining optimal dosage ranges for candidate molecules are alsoprovided. For example, the techniques may be used to identify a dosagerange that partially reduces transient tau protein phosphorylationduring stress, while greatly reducing permanent tau phosphorylation orinsoluble tau accumulation as a result of chronic stress conditions.

I. Tau Protein and its Signaling Pathway

Tau normally serves to bind and stabilize neuronal microtubules, tofacilitate their roles in cellular structure, polarity and transport(Stamer et al., 2002). Recent work suggests that tau plays a beneficalrole in supporting normal hippocampal memory-related function (Boekhoornet al., 2006). Phosphorylation can disrupt these activities and promotecytoskeletal destabilization (Sengupta et al., 1998). Aberrantlyphosphorylated forms of tau aggregate into PHFs, and these intoinsoluble NFTs, a defining feature of AD (Kopke et al., 1993). In thislight, the observation that acute stress results in tau-P at AD-relevantsites defines one potential means by which stress exposure may translateinto neuropathology. This effect has previously been reported a range ofrelatively strenuous challenge conditions, including heat shock(Papasozomenos, 1996), starvation (Yanagisawa et al., 1999; Planel etal., 2001, 2004), forced swimming in cold water (Korneyev et al., 1995;Korneyev, 1998; Okawa et al., 2003; Feng et al., 2005; Yoshida et al.,2006), glucoprivation (Planel et al., 2004), ether inhalation (Ikeda etal., 2007), and during hibernation (Arendt et al., 2003; Hartig et al.,2007). It is now common in the field of stress neurobiology todistinguish two broad categories of stress models. These may be termed“physiological” and “emotional”, and are distinguished by the nature ofthe sensory input that registers the challenge, the global pattern ofactivational responses they induce within the brain, the extent to whichthey invoke affective responses, and the circuitry that mediates certainadaptive responses to them (Sawchenko et al., 1996; Watts, 1996; Hermanand Cullinan, 1997; Dayas et al., 2001). Whereas the stressors shownpreviously to elicit stress-induced tau-P either are known, or mayreasonably be assumed, to fall in the physiological category, therestraint model employed herein is a prototypic emotional stressparadigm. Because established models of anxiety (elevated plus mazeexposure), fear (open field exposure, conditioned fear) and socialstress (social defeat, isolation), share with restraint such keyfeatures as a capacity to activate a stereotyped set of interconnectedcell groups in the limbic forebrain (Duncan et al., 1996; Campeau etal., 1997; Martinez et al., 2002), the present findings suggest that thegenerality of stress-induced tau-P may extend into the realm of lifestresses encountered in everyday experience.

Several stresses shown previously to elicit tau-P are associated withmarked reductions in body temperature (7-10° C.), which candifferentially modulate tau kinase and phosphatase activities, leadingto the hypothesis that hypothermia may be a common underlying mechanism(Planel et al., 2001; Planel et al., 2004). Restraint protocols like theone employed in the present study also result in reduced coretemperature, though of lesser magnitude (0.5-2° C.; Clement et al.,1989; Turek and Ryabinin, 2005; Meijer et al., 2006). It remains to bedetermined whether the strong correlation between body temperature andtau-P noted in other paradigms (Planel et al., 2007) extends torestraint and emotional stressors in general.

Tau isoforms are single gene products that differ by the inclusion ofinserts in an N-terminal projection domain and tandem repeats within aC-terminal microtubule-binding domain (Goedert et al., 1989). Whereashuman tau is normally phosphorylated at 2-3 moles/mole of protein,PHF-tau from AD brain is hyperphosphorylated at a 7-10 molar ratio(Kopke et al., 1993). Murine tau can be phosphorylated and form PHFs, invitro (Kampers et al., 1999). The inventors find that restraint provokesincreased phosphorylation at each of seven epitopes examined, all butone of whose responses is differentially modulated by CRFR status.Coupled with the finding that central CRF administration provokes PHF-1phosphorylation in a CRFR1-dependent manner, we suggest a mechanisminvolving mediation by CRFR1, which is normally restrained byCRFR2-based signaling.

Two aspects of our findings warrant further consideration. First,CRFR2-deficient mice displayed exaggerated responses to stress at fiveof seven phosphorylation sites and in three activated kinases. Whilesupporting an interaction with CRFR1 in regulating tau-P, the extent towhich these represent convergent or parallel effects is uncertain. Inpreliminary studies, we find that mice deficient in both CRFRs fail tomanifest acute restraint-induced tau-P, supporting some degree ofinterdependence (Rissman and Sawchenko, unpublished observations). Thisinterpretation is consistent with evidence that CRF, but not CRFR1expression or ligand binding, is upregulated in some brain regions ofCRFR2-deficient mice, and that CRFR1 antagonists can block aspects ofthe behavioral phenotype of these mutants (Kishimoto et al., 2000; Baleand Vale, 2003). These results suggest that exaggerated stress-inducedtau-P seen in CRFR2 mutants is not likely to result from alterations inCRFR1 expression or distribution. Second, while the consistent lack ofstress responsiveness of CRFR1 null mice may suggest a target forintervention in tau pathologies, this is offset by a tendency towardincreased basal levels of tau-P (AT8 and PHF-1 sites) and activatedkinase (notably JNK) expression in CRFR1 mutants. In addition, the needto supplement CRFR1-deficient mice with corticosterone during theperinatal period may complicate interpretation because it exposes theanimals to higher glucocorticoid levels during theirstress-hyporesponsive period (Baram et al., 1997) and the switch in thedominant form of tau from the 3-repeat to the 4-repeat isoform thatoccurs at this time (Kosik et al., 1989). It is noteworthy in bothrespects that acute pharmacological interference with CRF-R1 blockedrestraint-induced increments in tau-P without affecting basal levels,suggesting that the elevated baselines seen in knockouts may be asecondary or indirect consequence of chronic receptor deficiency.

Mice deficient in CRF-R1 exhibit impaired hormonal and behavioralresponses to stress, while CRF-R2 knockouts are over-reactive on many ofthe same measures (Smith et al., 1998; Timpl et al., 1998; Bale et al.,2000; Coste et al., 2000; Kishimoto et al., 2000). Subsequent workindicates that CRF-R interactions are not necessarily starklydifferential, but has generally supported convergent influences of thetwo receptor mechanisms on a range of stress-related endpoints (Bale andVale, 2004). CRF-R1 mRNA is prominently expressed in the pyramidal layerof Ammon's Horn, and in the hilar region of the dentate gyrus, whereasCRF-R2 transcripts are weakly expressed throughout the principal celllayers of both structures (Van Pett et al., 2000). Unfortunately, thelack of validated antisera has precluded decisive histochemicalcharacterization of receptor protein disposition. With respect toligands, it is unknown whether extrinsic CRF-containing inputs tohippocampus exist, leaving local interneurons (Chen et al., 2004) andthe cerebrospinal fluid (Arborelius et al., 1999) as potential sourcesfor delivery of CRF to hippocampal CRF-Rs. Among the urocortins, only avery sparse urocortin 1-immunoreactive input to hippocampus has beendocumented, likely originating from the midbrain Edinger-Westphalnucleus (Bittencourt and Sawchenko, 2000). Overall, while local receptormechanisms are in place, the sources of peptides in a position tointeract with them are unclear. The apparent paucity of CRF-R2 ligandsin hippocampal afferents suggests that their hypothesized interactionwith CRF-R1-dependent mechanisms may occur outside the hippocampus,proper.

Signaling Intermediates

Complementary monitoring of the activation state of tau kinasesidentified several candidate effectors. GSK-3β has been implicated incatalyzing tau-P at S₁₉₉, S₂₁₂, T₂₃₁, S₃₉6 and, to a lesser extent,S_(202/T205), and confers PHF-like changes (Liu et al., 2003). Itsactivity is stimulated or inhibited by phosphorylation at Y₂₁₆ and S₉,respectively (Cohen and Frame, 2001). Herein it is shown thatstress-induced increments in activated GSK-3β whose time course andgenotype dependence paralleled tau-P responses, without significantvariation in the inhibitory form (Okawa et al., 2003) or total(unphosphorylated) GSK3. The signaling intermediates that may link CRFRligand binding to alterations in GSK3 activity remain to be identified.Though commonly associated with a Gs-cAMP-protein kinase A mechanism,other signaling pathways can be activated downstream of CRFRs bydifferent G proteins in a cell type- and ligand-dependent manner(Grammatopoulos and Chrousos, 2002; Arzt and Holsboer, 2006; Hillhouseand Grammatopoulos, 2006). Contributing to the lack of clarity is alingering uncertainty as to the proximate mechanism of tyrosinephosphorylation of GSK, with some reports identifying this as anautophosphorylation event (Cole et al., 2004), and others implicatingdistinct tyrosine kinases, including Pyk2 and the src-related kinase,Fyn, in this regard (Lee et al., 1998; Hartigan et al., 2001).

In addition to GSK modulation, similar stress- and genotype-dependentchanges were observed in levels of activated JNK, implicated inmediating tau-P at the AT8 and PHF-1 epitopes (Atzori et al., 2001).High basal levels of phospho-JNKs in CRF-R1-deficient mice paralleled,and may explain, elevated AT8 and PHF-1 phosphorylation in this samegroup. The MAP kinases, ERK1 and 2, which can target all tau sitesexamined here except T231 (Drewes et al., 1992), were relativelyunresponsive. Relative levels of cdk5, considered a major tau kinaseactive at T231, AT8 and PHF-1 sites (Patrick et al., 1999), were alsostable across conditions, but one of its activator proteins, p35,exhibited strong CRFR-dependent stress responsiveness. In line withprevious findings (Okawa et al., 2003), we did not detect the truncatedp35 product, p25, which is a more potent cdk5 activator (Patrick et al.,1999).

Alterations in the activity of tau phosphatases, notably PP2A, have alsobeen implicated in stress-induced tau-P (Planel et al., 2001, 2004).Here we find increased relative levels of the catalytic subunit of PP2Aafter repeated stress, a finding that has been associated withdiminished enzymatic activity in vivo (Planel et al., 2001) and in vitro(Baharians and Schonthal, 1998), and attributed to a potentautoregulatory mechanism. Our failure to discern an acute stress effecton PP2A-c levels is not necessarily indicative of a lack of phosphataseinvolvement under these conditions.

Effects of Repeated Stress

Acute stress-induced tau-P has been consistently characterized as atransient and reversible phenomenon. As such, it would seem wellpositioned to contribute to the rapid dendritic/synaptic remodeling seenin the hippocampus in response to stress (Fuchs et al., 2006), but itsrelevance to longer-term pathogenesis has remained uncertain (see FIG.8). The present finding that repeated stress exposure results insustained elevations in tau-P, a portion of which is sequestered in adetergent-soluble form, supports such a potential, as the bulk ofdispersed PHFs from the AD brain reside in this same fraction (Iqbal etal., 1984; Rubenstein et al., 1986).

II. Amyloid Beta Pathology

The presence of amyloid plaques in hippocampus, neocortex, and amygdalais another major pathological hallmark of Alzheimer's disease. Theprinciple component of amyloid plaques is the amyloid beta (Aβ) peptide.Aβ is a peptide comprising 39 to 43 amino acids, processed from theamyloid precursor protein (APP), a transmembrane glycoprotein. APP isexpressed ubiquitously and is highly conserved in vertebrates. Aβpeptide is generated by the successive cleavage action of β and γsecretases. The β secretase cleaves at the carboxyl-terminal end of theAPP, whereas the γ secretase truncates APP at the amino-terminus. Thecleavage processing of the APP can generate a number of isoforms of Aβ,ranging from 39 to 43 amino acid residues in length. The most commonisoforms are 40 amino acids form (Aβ40) and 42 amino acids form (Aβ42).The Aβ42 form is the more fibrillogenic and is associated with diseasesgenesis.

Two lines of evidence relate APP processing to repeated stressparadigms. First, an isoform of Glycogen synthase kinase 3 (GSK-3α) hasbeen found to be required for maximal APP processing and Aβ production(Phiel et al., 2003). Both isoforms of GSK-3 are found to be able tophosphorylate tau. Although GSK-3β is more heavily studied in taupathology because of its localization as a component of NFTs and itspropensity to induce tangle pathology (Rankin et al., 2007), an analysisof kinase modulation indicated that GSK-3a is as potently activated byacute restraint as GSK-3β. In addition, the response of both isoformsshows a similar and differential dependence on CRFR status. Second, arecent report documented that restraint stress, or central CRFadministration, can increase interstitial fluid levels of Aβ□ albeit inAPP transgenic mice (Kang et al., 2007).

III. Modulation of CRF-R Signaling

A. Small Molecule Drugs

A wide array of CRF-R1 specific antagonist have been developed and areimplicated for the treatment of a number of disorders such as mooddisorders (affective disorders) and irritable bowel syndrome (see forexample, Tache et al., 2004). Preferably, a small molecule CRF-R1antagonist or CRF-R2 agonist of the invention is water soluble and maybe administered orally. Even more preferably, a small molecule for usein the invention is able to traverse the BBB.

A number of small molecule selective CRF-R1 antagonist are also known inthe art. For example, MTIP,3-(4-Chloro-2-Morpholin-4-yl-Thiazol-5-yl)-8-(1-Ethylpropyl)-2,6-Dimethyl-Imidazo[1,2b]Pyridazineis a CRF-R1 selective antagonist with no detectible activity on CRF-R2that has been implicated for the treatment of alcoholism.Advantageously, MTIP also has favorable solubility and has the abilityto cross the BBB (Gerhlert et al., 2007). Compounds like DMP696([4-(1,3-dimethoxyprop-2-ylamine)-2,7-dimethyl-8-(2,4-dichlorophenyl)-pyrazolo[1,5-a]-1,3,5-triazine]), DMP904([4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)-pyrazolo[1,5-a]-pyrimidine]) and derviatives are known to have >1,000 forselectivity for CRF-R1 relative to CRF-R2 (Li et al., 2005). Anothergroup of CRF-R1 antagonists includes but is not limited to CP-154,526and its methyl analog antalarmin (Keller et al., 2002).

Still other CRF-R1 antagonists contemplated for use herein areimidazolyl derivatives such as those described in U.S. Pat. No.7,125,990. For example, an orally active CRF-R1 selective antagonistR121919 (NBI 30775) has been developed to treat sleep disorders anddepression (Chen & Grigoriadis, 2005; Heinrichs et al., 2002). Stillfurther CRF-R1 antagonists contemplated for use herein include but arenot limited to R-278995, DMP-696, NBI 27914, NBI-35965, R121920,CRA1000, CRA1001, SSR125543A, DMP 695, DMP 904 and SN003 (McCarthy etal., 1999; Heinrichs et al., 2002; Million et al., 2003; Gilligan etal., 2000; Zorrilla et al., 2003; Holmes et al., 2003).

B. Peptide and Polypeptide Agonist and Antagonists

As discussed above, in some respects CRF-R agonists or antagonistcomprise CRF family members or derivatives thereof. For instance, amodified CRF family member may bind to CRF-R1, however not activate orminimally activate CRF-R1 signaling. In still further cases, a modifiedCRF peptide or polypeptide may bind to and agonize CRF-R2. As describedsupra, in preferred aspects a CRF-R agonist or antagonist for useaccording to the invention will be selective for CRF-R1 or CRF-R2 andhave little or no activity relative to the other receptor.

A variety of CRF family members are known that can be modified in orderto act as antagonists or antagonist. These molecules can be derived fromthe CRF family member of variety of organisms such as mice, rats, humansand frogs. Some non limiting examples of CRF family members includehuman and mouse Ucn 3 (SEQ ID NO:1 and SEQ ID NO:2) and human and mouseUcn 2 (SEQ ID NO:3 and SEQ ID NO:4). For example, the skilled artisanwill recognize that in some instances a human or murine Ucn 2 or Ucn 3may be used as a CRF-R2 agonist according to the invention. Furthermore,modifications of CRF family members may comprise amino acid deletions,amino acid insertions, amino acid substitutions and/or chemical changes,such as the insertion of lactam bridges, acetylation of amino acid sidechains or addition of PEG to the polypeptide. In general, modificationare made to accomplish one or more of the following; to alter CRF-Ractivation by the molecule, to enhance the molecules ability to blockCRF-R agonism or antagonism, to enhance CRF-R binding or selectivity ofthe molecule, to modify the pharmacokinetics of the molecule. Thus, itwill be understood that while any CRF family member can be modified inorder to generate a CRF-R agonist or antagonist, CRF family members withhigh affinity for CRF-R2 are preferred as CRF-R2 agonists.

It is also contemplated that in certain embodiments modified CRF familymember will preferentially agonize specific CRF-R2 protein isoforms. Forexample, the affinity of modified CRF family members for the alpha, betaand/or gamma protein isoforms for CRF-R2 can be assessed and CRF-R2agonists that are specific for one or more of the isoforms can beselected. This may be of particular advantage since it is known that theexpression of the various CRF-R2 isoforms is variable through-out thebody and thus by targeting specific CRF-R2 isoforms organs or tissuesexpressing that isoform (e.g., the CNS) may be more specificallytargeted. Again, this kind of specific CRF-R2 isoform targeting can bothincrease the efficacy and decrease potential side effects of CRF-R2agonists.

In additional aspects of the invention CRF polypeptides may be furthermodified by amino substitutions (e.g., to enhance selectivity for CRF-R1or CRF-R2) for example by substituting an amino acid at one or morepositions with an amino acid having a similar hydrophilicity. Theimportance of the hydropathic amino acid index in conferring interactivebiologic function on a protein is generally understood in the art (Kyte& Doolittle, 1982). It is accepted that the relative hydropathiccharacter of the amino acid contributes to the secondary structure ofthe resultant protein, which in turn defines the interaction of theprotein with other molecules, for example, enzymes, substrates,receptors, DNA, antibodies, antigens, and the like. Thus suchconservative substitution can be made in CRF-R antagonist or agonist andwill likely only have minor effects on their activity and ability tobind CRF-R. As detailed in U.S. Pat. No. 4,554,101, the followinghydrophilicity values have been assigned to amino acid residues:arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1);serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0);threonine (−0.4); proline (−0.5±1); alanine (0.5); histidine −0.5);cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8);isoleucine (−1.8); tyrosine (2.3); phenylalanine (−2.5); tryptophan(−3.4). These values can be used as a guide and thus substitution ofamino acids whose hydrophilicity values are within ±2 are preferred,those that are within ±1 are particularly preferred, and those within±0.5 are even more particularly preferred. Thus, any of the polypeptideCRF-R antagonist or agonist described herein may be modified by thesubstitution of an amino acid, for different, but homologous amino acidwith a similar hydrophilicity value. Amino acids with hydrophilicitieswithin +/−1.0, or +/−0.5 points are considered homologous.

It will also be understood that certain amino acids have specificproperties, and thus any amino acid substitution will abolish saidproperty. For example cysteine residues have the unique ability to formdisulfide bonds, that can be crucial for protein structure and activity.Thus, a substitution of cysteine residue for any other amino acid may beexpected, by one of skill in the art, to alter the activity of aprotein. Additionally, certain CRF-R agonists and antagonists are mayalso comprise a lactam bridge that structurally constrain thepolypeptide. Such lactam bridges can be formed between Glu and Lysresidues in a protein, and thus in certain cases amino acids may besubstituted for a Glu or a Lys in order to facilitated the insertion ofa lactam bridge. Such lactam bridges have been shown to be veryeffective in the generation of CRF-R2 binding peptides as described inRivier et al., 2002. Therefore in certain embodiments specific aminoacids may be substituted for unlike amino acids in order to facilitatethe insertion of an amino acid with a desired chemical or structuralproperty, such as a lactam bridge.

In certain embodiments of the current invention CRF-R agonists orantagonists comprise antibodies that bind to CRF-R2 or CRF-R1. CRF-Rantibodies may comprise polyclonal and/or monoclonal antibodies orfragments thereof. Methods for generating antibodies are well know tothose in the art. In general antibodies are raised against an antigenthat comprises at least a portion of a CRF-R amino acid sequence. Thusit will be understood that antibodies can be raised against the completeCRF-R amino acid sequence or portions thereof and that the amino acidsequence from any of the CRFR2 protein isoforms (alpha, beta and/orgamma) may be used as the immunogenic antigen. Furthermore, CRF-Rderived amino acid sequence may be further coupled to additional aminoacid sequences to increase its antigenicity.

In certain cases, CRF-R2 antibodies may bind preferentially to certainCRF-R2 protein isoforms (e.g. CRF-R2 alpha). In some preferred casesCRF-R2 antibodies can be made that bind to only one of the CRF-R2protein isoform. Such antibodies may have the advantage of being able totarget specific tissue and/or organs and therefore providing highlyspecific kinds of CRF-R2 agonists.

Not all antibodies that bind to CRF-R will act as agonists orantagonists thus in many cases the ability of an antibody to blockCRF-R1 agonism or mediate CRF-R2 agonism (e.g., an anti-idiotypicantibody) can be tested. Any of a variety of screening assays well knownin the art may be used to test CRF-R agonist or antagonist activity ofantibodies (e.g., see the binding assays described by Rivier et al.2002). Some specific methods for testing the efficacy of antibodyagonists/antagonists in vivo are described in examples provided here.

In certain further aspects of the invention CRF-R antibodies may bemodified to enhance their efficacy as CRF-R agonists/antagonists. Forexample, it is preferred that polypeptide therapeutics do not elicit animmune response. Thus, in the case where the subject for treatment is ahuman, antibodies may be human antibodies or humanized antibodies, so asto reduce the possibility of immune response. In yet furtherembodiments, it may be preferred that antibodies be single chainantibodies since the manufacture of single chain antibodies can besubstantially streamlined by production in insect or bacterialexpression systems. Thus, in certain cases, CRF-R antibodies that act asagonists or antagonists may be sequenced and the sequence used togenerate single chain antibodies.

It is additionally contemplated that nucleic acid aptamers that bind toCRF-R may be used to agonize or antagonize CRF-R activity. Methods forselecting aptamers by using recombinant CRF-R or fragments thereof topurify nucleic acid aptamers from a library, are well known in the art.The technique known as SELEX and can also be automated to enhance thespeed and efficacy of selection, for example see U.S. Pat. Nos.6,569,620 and 6,716,580. Aptamers identified to bind to CRF-R can thenbe screened for the ability to agonize or antagonize CRF-R1 or CRF-R2.In some specific cases, aptamers may be negatively selected using oneCRF-R protein (or protein isoform) and then positively selected using adifferent CRFR2 protein (or protein isoform) in order to identifyaptamers that specifically bind to particular CRF-R protein or isoforms.As used throughout the specification, “positive selection” meanscollecting molecules that bind to particular target, while “negativeselection” means collecting molecules that do not bind to a particulartarget. Aptamers according to this aspect of the invention may be DNA orRNA, and preferable comprise modified nucleotides that inhibitdegradation thereby enhancing activity.

Methods for synthesizing and purifying nucleic acids, such as CRF-Rbinding aptamers are well known to those in the art. For example DNAaptamers may be synthesized by PCR, while RNA aptamers can be generatedby in vitro transcription. In preferred embodiments, large scalepreparation of aptamers may be accomplished by chemical synthesis, thismethod allows for DNA, RNA and chemically modified oligonucleotides tobe incorporated into to the specific aptamer sequence.

IV. Therapeutic Compositions and Methods

Pharmaceutical compositions of the present invention comprise, in someinstances, an effective amount of a CRF-R1 antagonist and/or a CRF-R2agonist in a pharmaceutically acceptable carrier. The phrases“pharmaceutical or pharmacologically acceptable” refers to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, such as, forexample, a human, as appropriate. The preparation of a pharmaceuticalcomposition that contains CRF-R agonists/antagonists or additionalactive ingredient will be known to those of skill in the art in light ofthe present disclosure, as exemplified by Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, incorporated herein byreference. Moreover, for animal (e.g., human) administration, it will beunderstood that preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents, targetingagents (e.g., CNS targeting agents), lubricants, sweetening agents,flavoring agents, gels (e.g., gelatin), dyes, such like materials andcombinations thereof, as would be known to one of ordinary skill in theart (see, for example, Remington's Pharmaceutical Sciences, 18th Ed.Mack Printing Company, 1990, pp. 1289-1329, incorporated herein byreference). Except insofar as any conventional carrier is incompatiblewith the active ingredient, its use in the therapeutic or pharmaceuticalcompositions is contemplated.

A therapeutic composition of the invention may comprise different typesof carriers depending on whether it is to be administered in solid,liquid or aerosol form, and whether it need to be sterile. The presentinvention can be administered intravenously, intradermally,intraarterially, intraperitoneally, intracranially, mucosally,intraocularally, subcutaneously, or intranasally, intravitreally,intravaginally, intrarectally, topically, intrathecally,intracerebroventricularly, orally, locally (e.g., into the CNS), viainhalation (e.g., aerosol inhalation), via a lavage, in cremes, in lipidcompositions (e.g., liposomes), or by other method or any combination ofthe forgoing as would be known to one of ordinary skill in the art (see,for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack PrintingCompany, 1990, incorporated herein by reference).

The actual dosage amount of a composition of the present inventionadministered to a subject can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated (i.e., type of tauoopathy), previous orconcurrent therapeutic interventions, idiopathy of the patient and onthe route of administration. The practitioner responsible foradministration will, in any event, determine the concentration of activeingredient(s) in a composition and appropriate dose(s) for theindividual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof. In the case, ofproteinacious compositions of the invention is may also be preferablethat the action of proteases be inhibited during storage ofcompositions. This can be accomplished by the additional of proteaseinhibitors and/or the storage of the compositions at low temperatureprior to administration.

In embodiments where compositions according to the invention areprovided in a liquid form, a carrier can be a solvent or dispersionmedium comprising but not limited to, water, ethanol, polyol (e.g.,glycerol, propylene glycol, liquid polyethylene glycol, etc.), lipids(e.g., triglycerides, vegetable oils, liposomes) and combinationsthereof. The proper fluidity can be maintained, for example, by the useof a coating, such as lecithin; by the maintenance of the requiredparticle size by dispersion in carriers such as, for example liquidpolyol or lipids; by the use of surfactants such as, for examplehydroxypropylcellulose; or combinations thereof such methods. In manycases, it will be preferable to include isotonic agents, such as, forexample, sugars, sodium chloride or combinations thereof

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In certain embodiments, an oral composition may comprise one or morebinders, excipients, disintegration agents, lubricants, flavoringagents, and combinations thereof. In certain embodiments, a compositionmay comprise one or more of the following: a binder, such as, forexample, gum tragacanth, acacia, cornstarch, gelatin or combinationsthereof; an excipient, such as, for example, dicalcium phosphate,mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate or combinations thereof a disintegratingagent, such as, for example, corn starch, potato starch, alginic acid orcombinations thereof a lubricant, such as, for example, magnesiumstearate; a sweetening agent, such as, for example, sucrose, lactose,saccharin or combinations thereof a flavoring agent, such as, forexample peppermint, oil of wintergreen, cherry flavoring, orangeflavoring, etc.; or combinations thereof the foregoing. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, carriers such as a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both.

Additional formulations which are suitable for other modes ofadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum, vagina or urethra. After insertion, suppositoriessoften, melt or dissolve in the cavity fluids. In general, forsuppositories, traditional carriers may include, for example,polyalkylene glycols, triglycerides or combinations thereof. In certainembodiments, suppositories may be formed from mixtures containing, forexample, the active ingredient in the range of about 0.5% to about 10%,and preferably about 1% to about 2%.

A. Dosages

CRF-R agonists/antagonists of the invention will generally be used in anamount effective to achieve the intended purpose. For use to treat ordelay the onset or progression of a tauopathy, the molecules of theinvention, or pharmaceutical compositions thereof, are administered in atherapeutically effective amount. A therapeutically effective amount isan amount effective to ameliorate or prevent the symptoms, or onset orprogression of clinical disease of, the subject being treated.Determination of a therapeutically effective amount is well within thecapabilities of those skilled in the art, especially in light of thedetailed disclosure provided herein. For example, as described supra, incertain instances an effective amount of a compound of the invention maybe defined by the ability of the compound to prevent a given amount ofstress-induced tau phosphorylation.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat includes the IC₅₀ as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art and the specifictechniques described herein. One having ordinary skill in the art couldreadily optimize administration to humans based on animal data.

The amount of molecules administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

The therapy may be repeated intermittently while symptoms detectable oreven when they are not detectable (e.g., to prevent to onset ofsymptoms). The therapy may be provided alone or in combination withother drugs. In the case of AD, the drugs that may be used incombination with Aβ vaccines (e.g., see U.S. Pat. No. 6,787,140 and PCTpublication, WO 2005/014041) an acetylcholinesterase inhibitor such asDonepezil, Rivastigmine or Galantamine, Vitamin E, or ananti-inflammatory drug such as a nonsteroidal anti-inflammatory drug(NSAID) (De La Garza, 2003). Non-limiting examples NSAIDs include,ibuprofen, ketoprofen, piroxicam, naproxen, naproxen sodium, sulindac,aspirin, choline subsalicylate, diflunisal, oxaprozin, diclofenac sodiumdelayed release, diclofenac potassium immediate release, etodolac,ketorolac, fenoprofen, flurbiprofen, indomethacin, fenamates,meclofenamate, mefenamic acid, nabumetone, oxicam, piroxicam, salsalate,tolmetin, and magnesium salicylate.

Methods for estimating dose conversions between animal models and humanshave previously been developed. In general these algorithms have beenused to extrapolate an animal dose to a dose that would be tolerated bya human. For example, methods for dose conversion have previously beendisclosed by Freireich et al. (1966). The conversion methods taught byFreireich calculate equivalent doses between species using surface area(m²) rather than mass (kg), a method that correlates much more closelyto actual data than body mass conversions. Specifically, Freireichteaches how to use an animal 10% lethal dosage (LDio) value to estimatethe maximum tolerated doses in a human. Freireich also discussed methodfor converting a dose in mg/kg to a dose in mg/m² by using the “km”conversion factor for the given animal. For example, in the case of alaboratory mouse the km is approximately 3.0. Thus, in mice mg/m²=km(3.0 for mice) X dose in mg/kg.

More recent studies regarding species dose scaling have furtherelaborated upon the methods of Freireich. These newer studies havereduced error associated with conversion between species to determinehuman tolerable doses. For example, Watanabe et al. (1992) describesthat a conversion of doses between species using body surface area maynot be the most accurate method per se for predicting a human equivalentdosage. Nonetheless, the scaling factors set forth by Watanabe yieldresults that are with-in the margin of error of the older Freireichconversions. Currently accepted methods for determining a properstarting dose in humans expand upon the methods set forth by Freireich.For example, Mahmood et al. (2003) provides a discussion regarding thechoice of a proper starting dose in humans given dose studies inanimals.

B. Toxicity

Preferably, a therapeutically effective dose of CRF-R agonist orantagonists described herein will provide therapeutic benefit withoutcausing substantial toxicity.

Toxicity of the molecules described herein can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., by determining the LD₅₀ (the dose lethal to 50% of the population)or the LD₁₀₀ (the dose lethal to 100% of the population). The dose ratiobetween toxic and therapeutic effect is the therapeutic index. Proteinswhich exhibit high therapeutic indices are preferred. The data obtainedfrom these cell culture assays and animal studies can be used informulating a dosage range that is not toxic for use in human. Thedosage of the proteins described herein lies preferably within a rangeof circulating concentrations that include the effective dose withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage form employed and the route of administration utilized.The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. (See,e.g., Fingl et al., 1975).

C. CNS Targeted Therapy

In certain aspects of invention concerns compositions such as CRF-R1antagonists and/or CRF-R2 agonists that are CNS targeted. A variety ofmolecules are known to confer CNS targeting. For instance, certainantibodies are know to cross the BBB, thus such antibodies may be usedto transport a payload, such as a CRF-R agonist/antagonist to the CNS.Some specific antibodies that may be used include but are not limited toantibodies to transferrin receptors (e.g., OX26) or antibodies to theinsulin receptor (Schnyder & Huwyler, 2005). Other polypeptides may alsobe used to target the CNS such as cationized albumin. Thus, polypeptideCNS targeting agents may in some aspects, be bound to a CRF-R agonist orantagonist for use according to the invention. In some very specificcases, a peptide (or polypeptide) CRF-R agonist or antagonist may beprovided as a fusion protein with a CNS targeting polypeptide. In stillother cases nanoparticles such as Polysorbate 80-coatedpolybutylcyanoacrylate nanoparticles may be used to deliver compositionsto the CNS (Olivier, 2005). In still further aspects, CNS targetingpolypeptides may be conjugated to liposomes to form CNS targetingcomplexes (Schnyder & Huwyler, 2005). Furthermore, peptide andpolypeptide CRF-R1 antagonists and/or CRF-R2 agonists may be targeted tothe CNS by glycosylation, for example as described in Egleton & Davis(2005). In yet further aspects, viral vectors may be used to targeteddelivery of peptides or polypeptides to the CNS. For example, lentiviralvector systems for polypeptide delivery are known in the art, see forexample Spencer & Verma (2007).

EXAMPLES

The following examples are included to further illustrate variousaspects of the invention. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples that followrepresent techniques and/or compositions discovered by the inventor tofunction well in the practice of the invention, and thus can beconsidered to constitute preferred modes for its practice. However,those of skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentswhich are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

Example 1 Experimental Methods

CRF-R Knockout Mice

Mutant mice and littermate wild type (wt) controls were bred fromheterozygote breeder pairs of established lines derived from backcrossesof founder mice to achieve a pure C57BL/6 background (Smith et al.,1998; Bale et al., 2000). Genotyping was performed by PCR analysis oftail DNA, and appropriate males were used for experimentation at 15-22weeks of age. Pregnant females bearing fetuses carrying a mutant CRF-R1allele received drinking water with 10 μg/ml corticosterone from daysE12-P14 to prevent early mortality due to pulmonary dysplasia (Smith etal., 1998). Because CRF-R1−/− mice exhibit adrenal cortical agenesis,experimental animals were reinstated on corticosterone supplementationfor 21 days prior to testing to allow the normal nocturnal bias inappetitive behavior to approximate the circadian fluctuation incirculating hormone levels. Blood samples were collected at the time ofsacrifice and plasma corticosterone levels determined by RIA to assessthe effectiveness of the replacement regimen. The Institutional AnimalCare and Use Committee of the Salk Institute approved all experimentalprotocols.

Adrenalectomy (ADX) and Corticosterone Replacement

8-12 week old male wt C57BL/6 mice (Jackson Labs, Bar Harbor, Me.)underwent ADX via bilateral incisions on the dorsolateral flanks underisoflurane anesthesia (to effect; 3-5% vapor/total vol of 02). ADX micereceived replacement corticosterone (10 μg/ml; Sigma, St. Louis, Mo.) indrinking water immediately after surgery. Animals were utilized instress experiments 21 d after surgery.

Restraint Stress

Acute restraint stress involved placing mice in ventilated 50 ml conicaltubes for 30 min; repeated stress involved 14 consecutive dailyexposures. Animals were killed at various intervals ranging from 20 minto 24 hr after stress. Control mice were handled comparably, but werenot otherwise manipulated.

Intracereboventricular Injections

CRF-R1−/− (U.S. Pat. No. 6,147,275) and CRF-R2−/− (U.S. Pat. No.6,353,152) mice, along with age-matched wt controls (n=3/group), wereanesthetized with isoflurane, and implanted stereotaxically with 26-gaguide cannulae (Plastics One, Wallingford, Conn.) aimed to terminateabove the lateral ventricle. Cannulae were affixed to the skull withdental acrylic adhering to jeweler's screws partially driven into theskull, and sealed externally with stylets. After 7 d recovery, styletswere replaced with 33-ga injection cannulae, and 2 hr later the animalswere remotely injected with 0.5 μg synthetic mouse/human CRF in 2 μlsaline, or vehicle alone, over ˜1 min. To approximate the time frameused in acute stress experiments, animals were killed 40 min after icyinjection and perfused for immunohistochemistry, as above. CRF wasgenerously provided by Dr. J. Rivier (Salk Institute).

In Vivo Pharmacology

The small molecule, CRF-R1-selective antagonist, antalarmin (Webster etal., 1996), was administered at 20 mg/kg by intraperitoneal (ip)injection 20 min before stress exposure. All animals were handled twicedaily for 28 d prior to experimentation, and received daily mock ipinjections to minimize stress of injection at testing. Antalarmin wassolubilized in equal volumes of absolute ethanol and Cremaphor EL(Sigma-Aldrich, St. Louis, Mo.), using previously described protocols(Webster et al., 1996; Pernar et al., 2004). This stock solution wasdiluted in prewarmed (50° C.) distilled water and adjusted to a finalconcentration of 4 mg/ml immediately prior to injection.

Western Blot Analysis

Mice were deeply anesthetized with sodium pentobarbital (40 mg/kg),which has been demonstrated to not influence the phosphorylation stateof tau over the time frame employed here (Papasozomenos, 1996). Aftersedation, the animals were decapitated and the hippocampus was rapidlydissected and frozen on dry ice. Hippocampal tissues were homogenized inRIPA buffer (50 Mm Tris-HCl pH 7.4, 0.1% SDS, 1% NP40, 0.25% sodiumdeoxycholate, 150 nM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM Na₃VO₄, and 1 μMokadaic acid). Immediately before homogenization, protease inhibitorsPMSF, NaF (1 mM), aprotinin, leupeptin, pepstatin (1 μg/ml each) wereadded. RIPA fractions were obtained by centrifuging twice at 40,000 gfor 20 min, and the supernatant was collected. For analysis of tausolubility (repeated stress), sequential fractionation of RAB and RIPAextracts were performed as described (Higuchi et al., 2002; Kraemer etal., 2003). In this case, tissues were first homogenized in high-saltRAB (0.1 M IVIES, 0.75 NaCl, 1 mM EGTA, and 0.5 mM MgSO₄) and thencentrifuged at 40,000 g for 40 min. The resultant supernatant wascollected (soluble RAB fraction). Resultant pellets were resuspended inRIPA buffer as described above to obtain detergent-soluble fractions.Protein concentrations were determined using a BCA Protein Assay Kit(Pierce Biotechnology, Rockford, Ill.). Proteins were then boiled insample buffer containing SDS, 2-mercaptoethanol, and glycerol at 95° C.for 5 min. 6 μg of protein was then loaded and electrophoreticallyseparated on a 12% SDS-polyacrylamide gel. Proteins were transferred tonitrocellulose membrane (0.2 μm, BioRad, Hercules, Calif.) and incubatedin primary antibodies diluted in 5% milk-PBS-T overnight at 4° C.Primary antibodies were detected with either anti-mouse or rabbithorseradish peroxidase-linked secondary antibodies (1:1000 Calbiochem,San Diego, Calif.) and developed with an enhanced chemiluminescenceWestern blot detection kit (Supersignal West Pico, PierceBiotechnology). Background subtraction was performed and quantitativeband intensity readings were obtained using NIH Image software.

Antibodies

Well-characterized phospho-specific antibodies were used for detectionof several phosphorylated residues on mouse tau. For Western blots,T₁₈₁, S₁₉₉, S₂₁₂, T₂₃₁, S₄₂₂ (1:1000, Biosource), S²⁰²/T²⁰⁵ (1:500, AT8,Pierce Biotechnology), S^(396/404) (1:1000, PHF-1, gift from Dr. P.Davies). These antibodies were chosen based on their ability to resolvetarget bands at the appropriate molecular weight for phosphorylated tau(i.e., ˜50-75 kDa). Phospho-specific antibodies against S²¹⁷, S²⁶², S³⁵⁶and S⁴⁰⁹ phosphorylated tau (Biosource) were also tested.Immunohistochemical analyses used PHF-1 for detection of phosphorylatedtau. Specificity of PHF-1 for phosphorylated tau in mouse tissue wasconfirmed by pretreating sections from stressed mice with alkalinephosphatase (40 mg/ml, Sigma-Aldrich). This treatment eliminateddetectable PHF-1 labeling in all experimental groups. For assessment oftau kinases, specific antibodies either to phosphorylation sites oractivator proteins were used. For glycogen synthase kinase (GSK-3),total GSK-3β (1:2500, BD Transduction Labs, San Diego, Calif.),activated GSK-3β (pY216, 1:1000, BD Transduction Labs), inactive GSK-3β(pS9, 1:1000, Cell Signaling), cyclin-dependent kinase 5 (cdk-5; 1:1000;Calbiochem, San Diego, Calif.), cdk5 activator proteins, p25 and p35(1:1000, Santa Cruz Biotechnology, Santa Cruz, Calif.), phosphorylatedc-Jun-N-terminal kinase (JNK, 1:1000; Cell Signaling, Danvers, Mass.),mitogen activated protein kinases (ERK 1/2, 1:500, Cell Signaling), thecatalytic subunit of protein phosphatase 2A (PP2A-c, 1:5000, BDTransduction Labs). β-actin (1:2000, Sigma-Aldrich) was used as acontrol for protein loading.

Immunohistochemistry

Mice were perfused with 4% paraformaldehyde, as previously described(Bittencourt and Sawchenko, 2000). 30 μm-thick frozen sections were cuton a sliding microtome and stored at −20° C. in cryoprotectant solution(20% glycerol and 30% ethylene glycol in 0.1 M phosphate buffer) untiluse. Free-floating sections containing the hippocampus were used todetect tau-P with PHF-1 (1:500) in mouse tissues using Mouse-on-MouseImmunodetection Kit reagents (Vector, Burlingame, Calif.) to avoiddetection of endogenous mouse immunoglobulin. Endogenous peroxidase wasquenched with 0.3% hydrogen peroxide, followed by 1% sodium borohydrideto reduce free aldehydes. Reaction product was developed using anickel-enhanced glucose oxidase method (Shu et al., 1988).

Statistical Analyses

Integrated intensity readings from Western blots were analyzed usingeither a one- or two-way ANOVA using Prism4 Software (GraphPad, SanDiego, Calif.). Resultant data were plotted on bar graphs, with dataexpressed as mean±SEM percentage of control values.

AD Transgenic Mice

The PS/APP mouse line was originally purchased from another researchlaboratory. These mice express the Swedish familial AD mutation of humanamyloid precursor protein (APP) (APPK670N,M671L; line Tg2576) and thefamilial AD mutation of presenilin-1 (PSEN1-dE9) (Jankowsky et al.,2001). Hemizygous double mutant PS/APP offspring and non-transgenic micewere used in experimentation, as it has been reported that homozygotelines of Tg2576 cannot be generated (McGowan et al., 1999). Genotypeswere determined by PCR analysis of ear or tail DNA.

Lipopolysaccharide (LPS) Injection Induced Physiological Stress

Animals adapted to handling and intraperitoneal (ip) injection wereadministered LPS from Salmonella typhimurium (Sigma-Aldrich, St. Louis,Mo.) at 10 μg/kg, ip in 100 μl sterile saline. Control animals werepretreated similarly and received injections of vehicle.

Systemic Antagonist Administration

For acute stress experiments, CRFR1-selective small molecule antagonist,antalarmin (Webster et al., 1996), was administered at 20 mg/kg byintraperitoneal (ip) injection 20 min before stress exposure. Allanimals were handled twice daily for at least 14d prior toexperimentation, and receive daily mock ip injections to minimize stressof injection at testing. Antalarmin was solubilized in equal volumes ofabsolute ethanol and Cremaphor EL (Sigma-Aldrich, St. Louis, Mo.), asdescribed (Webster et al., 1996; Pernar et al., 2004). This stocksolution was diluted in prewarmed (50° C.) distilled water and adjustedto a final concentration of 4 mg/ml immediately prior to injection. Forchronic administration, antalarmin was administered twice daily via ipinjection (Wong et al., 1999).

Chronic Variable Stress (CVS)

CVS involved daily exposure to either two brief or one sustainedstressor over 15d. Brief stressors included elevated plus maze exposure(30 min), shaker stress (30 min on an orbital shaker at 100 rpm), iphypertonic saline injection (0.4 ml of 1.5M saline), restraint (30 min),tail suspension (10 min) and forced swimming (10 min in RT water).Sustained stressors included social isolation (single housing for 1 d),wet bedding (1 d), and cold exposure (8 hr at 5-7° C.). Stressors werepresented at unpredictable times of day and in random sequence.

Electron Microscopy

Negative staining and immunogold labeling was carried out in extractsdeposited on carbon-coated grids (400 m, Pelco, Clovis, Calif.), whichhas been exposed to glow discharge for 5 min. At each step, excessliquid was removed by wicking with bibulous filter paper. Grids wereplaced sequentially on drops of reagents spotted on parafilm. Except forthe samples, all reagents were 0.2 μm filtered. The grids were placed on10 μl of a sample for 90 seconds. The grids were then blocked with 2%BSA, 0.1% fish gelatin in KPBS for 10 minutes, which was followed by atreatment with 20 μl primary antibody (e.g., anti-PHF-1 at 1:100) inblocking buffer for overnight at 4° C., in a sealed, humidified chamber.The next day, the grids were washed 3 time for 5 minutes each inblocking buffer, and placed on 20 μl secondary antibody at 1:50 (goatanti-mouse IgG coupled to 10 nm gold particles, BioCell, RanchoDominguez, Calif.) for 2 hr at room temperature. The grids were thenwashed 3 times for 5 minutes each in blocking buffer, and washed 3 timesfor 5 minutes in water. Following the wash, the grids were stained for60 seconds with 2% phosphotungstic acid pH 7.0 or 4% uranyl acetate, anddried with filter paper, followed by an air dry treatment. After thepreparation, the samples were examined in a JEOL 100 CX II transmissionelectron microscope (JEOL USA, Inc., Peabody, Mass.).

ELISA

Soluble, detergent-soluble, and formic acid soluble fractions wereextracted from stressed mice for ELISA assay for Aβ using reagentsavailable in kit form (Biosource mouse (3-amyloid colometric immunoassaykit, Invitrogen, Carlsbad, Calif.). Extract preparation is detailedabove. Formic acid extraction involved solubilization ofdetergent-insoluble pellets with 70% formic acid and subsequentcentrifugation at 40,000 g for 20 min. Data were read at 450 nm using aBioRad Lumimark Plus Microplate reader (BioRad, Hercules, Calif.)attached to a PC computer with Microwin software (MicroWin A G,Wallisellen, Switzerland).

Example 2 Tau Protein Phosphorylation after Acute Restraint Stress

To determine whether increased tau phosphorylation (tau-P) wasobservable in response to acute restraint, an acknowledged “emotional”stressor (Sawchenko et al., 2000). Western blot analysis was used toexamine tau phosphorylation at several AD-relevant N- and C-terminalsites (S¹⁸¹, S¹⁹⁹, S²⁰²/T²⁰⁵ (AT8), T²¹², T²³¹, S^(396/404) (PHF-1), andS⁴²²) in hippocampal extracts from C57BL/6 mice sacrificed at variousintervals after a single 30-min episode of restraint stress. Relative tobasal (non-stressed) values, all sites exhibited significant increasesin phosphorylation that were apparent at the termination of stress (0min), with peak elevations (2-10 fold) achieved 20-40 min later andsustained through 60 min (FIG. 1). By 90 min, levels were reduced to ornear those of unstressed controls. These results demonstrate that arepresentative emotional stressor induces rapid and reversible increasesin tau-P at multiple AD-relevant sites. Increments in tau-P were quitestable over 20-60 min post-stress, and the 20 min time point wasselected for use in subsequent analyses.

Example 3 Glucocorticoid Involvement in Stress-Induced TauPhosphorylation

Because of the dominant role of glucocorticoids in mediating stresseffects on the CNS and their implication in AD neuronal damage (Sapolskyet al., 1985, 1986) and in mouse models of AD (Green et al., 2006),studies were undertaken to determine whether stress-induced tau-P wasdependent on stress-induced glucocorticoid secretion. Although a priorstudy employed immunoassay and found no effect of adrenalectomy (ADX) onPHF-1 reactivity after acute cold water stress (Korneyev et al., 1995),phosphatase inhibitors were not utilized and only soluble fractions oftau proteins were examined. Studies described here examinedstress-induced tau-P responses at the AT8 and PHF-1 sites in extracts ofhippocampus from ADX and control mice exposed to acute restraint stress(FIG. 2A-B). ADX animals received replacement corticosterone in drinkingwater (10 μg/ml) to approximate basal hormone titers and their diurnalvariation. Robust stress-induced tau-P responses were observed in ADXmice that did not significantly differ from those of intact controls ateither the AT8 and PHF-1 phosphorylation sites (each P>0.10). Theseresults confirm that acute stress-induced tau-P is not dependent uponglucocorticoid secretion.

Example 4 CRF-Rs Differentially Regulate Stress-Induced TauPhosphorylation

The CRF family of signaling molecules is broadly involved inphysiological and behavioral responses to stress (Chadwick et al.,1993), and is altered early in AD progression (Davis et al., 1999).However, the nature of its involvement in AD neuropathology is unclear.In view of this deficiency, the role of CRF-Rs in acute stress-inducedtau phosphorylation was investigated using mice deficient in CRF-R1(Smith et al., 1998; U.S. Pat. No. 6,147,275) or CRF-R2 (Bale et al.,2000; U.S. Pat. No. 6,353,152). Western analyses (FIG. 3A-B) indicated atendency for CRF-R1−/− mice to exhibit higher basal levels of tau-P thanunstressed wild type (wt) animals at several sites, although thisdifference was statistically reliable only at the AT8 epitope (P<0.01).More importantly, CRF-R1-deficient mice failed to exhibit significantincreases in tau-P at any site at 20 min after stress, compared toage-matched wt controls. By contrast, CRF-R2 knockouts displayed normalbasal levels of tau-P (P>0.05 vs wt), but showed robust responses toacute stress that commonly exceeded those seen in wt animals.Specifically, phosphorylation responses of CRF-R2−/− mice weresignificantly greater that those of wt animals at the S¹⁸¹ (P<0.01),S¹⁹⁹ (P<0.001), T²¹² (P<0.05), T²³¹ (P<0.05), and PHF-1 (P<0.01) sites.Phosphorylation responses of CRF-R2 null mice at the AT8 and S422 sitesdid not differ significantly from those of wt stressed animals (P>0.05).

To probe the localization of tau-P responses, immunohistochemicalmethods were used to examine the distribution of PHF-1 phosphorylation,and its stress and CRF-R-dependence. Immunolabeling results were highlycompatible with biochemical data in showing prominent upregulation ofPHF-1 staining in wt mice in response to stress, which was attenuatedand exaggerated in CRF-R1- and CRF-R2-deleted animals, respectively(FIG. 4A). In the dentate gyrus of stressed wt animals, PHF-1 positivecell bodies were seen primarily in the hilus (polymorph and subgranularregions), but also in deep aspects of the granule cell layer. PHF-1positive mossy fibers and a band of punctate (presumably axonal)elements in the inner third of the molecular layer was also observed. InAmmon's Horn, dominant features included labeled perikarya scatteredmainly throughout the pyramidal layer and proximal dendritic zones, andbands of axon terminal-like puncta engulfing the pyramidal cell layerand in stratum lacunosum-moleculare. Radially oriented processes, sometraceable to labeled cell bodies and presumably representing dendriticlabeling, were seen in stressed wt and CRF-R2-deficient animals.Alterations in immunostaining as a function of stress and genotype weremanifest as differences in the number and/or intensity of labeledelements, with no discernible differences in distribution.

To determine whether CRF is capable of independently elicitinghippocampal tau-P, wt and CRF-R-deficient mice were implanted withlateral ventricular cannulae for icy injection. Resultant PHF-1immunoreactivity was examined 40 min after administration of CRF (0.5 μgin 5 μl) or vehicle (FIG. 4B). The general pattern of results wassimilar to that observed in response to stress, in that wt and CRF-R2−/−mice treated with peptide displayed robust increases in hippocampaltau-P, while labeling in CRF-R1−/− mice was comparable to the low levelseen in saline-injected controls. In the dentate gyrus, PHF-1 positivecell bodies were seen in the hilus and deep aspects of the granule celllayer (FIG. 4B). In Ammon's Horn, PHF-1 labeling was observed in mossyfibers and in the form of punctate pericellular labeling throughout thepyramidal cell layer and, more sporadically, in the dendritic zone, withparticular concentration at the septal pole of the hippocampus. Incontrast to stress effects, however, no labeling of pyramidal neurons ortheir processes was observed after icy CRF injections. These findingsindicate that central CRF administration at least partiallyrecapitulates the effects of stress, and demonstrates a similardependence on CRF-R integrity.

Example 5 Effects of Pharmacologic Blockade of CRF-R1

Interpretation of data derived from conventional knockout animals may becomplicated by developmental or indirect effects of lifelong lack ofexpression of the targeted gene. This is particularly true ofCRF-R1-deficient mice, which exhibit chronically impairedpituitary-adrenal function (Smith et al., 1998). Despite efforts tomitigate such effects by steroid replacement perinatally and immediatelyprior to experimentation (see Example 1), confidence in the assertion ofa regulatory role for CRF-R1 in this context would be bolstered if theeffects were maintained in the face of acute disruption of receptorfunction. Therefore, the effect of antalarmin, a small molecule,selective CRF-R1 antagonist (Webster et al., 1996), on basal andstress-induced tau-P was studied. Neither antalarmin nor the vehicleused for its administration significantly altered basal levels of tau-Pat the AT8 or PHF-1 sites, relative to untreated controls (FIG. 5).However, antalarmin treatment prevented stress-induced increments inphosphorylation at both sites (lanes 5-6; each P>0.10 vs. untreatedcontrols). Phosphorylation responses in stressed, vehicle-treatedanimals were comparable to those of stressed, untreated controls, andsignificantly elevated over vehicle control levels (P<0.01). Thesefindings support a specific involvement of CRF-R1 signaling instress-induced tau-P.

Example 6 Modulation of Tau Kinase Activity by Stress and CRF-Rs

To identify potential mediators of acute stress-induced tau-P,antibodies specific to active and inactive states of kinases implicatedin tau-P in same tissue extracts were employed to interrogate changes instress-induced kinase activation, and their CRF-R dependence (FIG. 6).Several kinases examined, including the active (phosphorylated at Y²¹⁶,or pY²¹⁶), but not the inactive (pS⁹), or total (unphosphorylated) formof glycogen synthase kinase-3β (GSK-3β), the pT¹⁸³/Y¹⁸⁵ form of the 46and 54 kDa c-Jun N-terminal protein kinases (JNK46/54), and thepT²⁰²/Y²⁰⁴ form of the mitogen-activated protein kinases, ERK2, but notERK1, displayed upregulation in response to acute restraint. The timecourses of stress effects seen on these kinases were similar to thoseshown in FIG. 1 for stress-induced tau-P. Relative levels ofcyclin-dependent kinase 5 (cdk5) were unchanged from steady state overthe post-stress intervals examined, but one its regulatory proteins,p35, was robustly upregulated. Despite this change in p35, its truncatedproduct, p25 was not reproducibly detected.

When tested in hippocampal extracts from wt and knockout mice, each ofthe stress-responsive kinase forms or regulators also exhibitedmodulation as a function of CRF-R status that mirrored some or all ofeffects of genotype on restraint-induced tau-P. The activated (pY²¹⁶)form of GSK-3β, implicated in phosphorylating tau at S¹⁹⁹, S²¹², T²³¹and PHF-1 sites, was most similar in that the stress-induced incrementseen in wt mice was not evident in CRF-R1−/− animals, and exaggerated inCRF-R2−/− mice. Phosphorylation responses of both JNK isoforms were alsosignificantly greater in CRF-R2 knockouts than in wt controls (P<0.05).These kinases also exhibited pronounced elevations in basalphosphorylation in CRF-R1-deficient mice, whose magnitude rivaled orexceeded stress-induced levels in wt mice. This may relate to theelevated tau-P levels seen under this condition at the AT8 and PHF-1sites, though less marked elevations of phosphorylated GSK-313 and ERK2,and of p35 levels, in unstressed CRF-R1−/− mice may also contribute inthis regard. Overall, these results identify several tau kinases aspotential effectors of CRF-R-dependent effects of acute emotional stresson tau-P.

Example 7 Tau Phosphorylation in Response to Repeated Stress

Because past and present data characterize acute stress-induced tau-P asa transient phenomenon, its relevance to neuropathology may bequestioned. Data from animal models and the AD brain have demonstratedthat NFTs and other manifestation of tau pathology are dependent onaberrantly phosphorylated tau being sequestered into insoluble cellularfractions (Iqbal et al., 1994). Therefore, sequential fractionation wasused in the absence (RAB buffer; see methods) and then in the presenceof detergents (RIPA buffer) to compare the persistence and solubility ofphosphorylated tau in animals subjected to acute versus repeated (14consecutive daily exposures) restraint stress (FIG. 7). Groups of micein each condition were sacrificed 20 min or 24 hr after their final oronly stress episode. Results from the acute stress condition replicatedfindings detailed above in showing increased phosphorylation at the AT8and PHF-1 sites 20 min post-stress. At 24 hr post-stress, relativelevels of tau-P were indistinguishable from unstressed animals (lane 3).In terms of solubility, phosphorylated tau induced by acute stress wasdetected only in the soluble fraction; that is, no additional signal wasevident upon further extraction with detergent (lanes 6-8). In contrast,under repeated stress conditions, comparably elevated AT8 and PHF-1signal were present in soluble fractions at both 20 min and 24 hr afterthe final restraint episode. In addition, extraction ofdetergent-soluble proteins (RIPA) revealed significant occurrence ofphosphorylated tau at both time points (lanes 9-10). These resultssuggest that repeated stress leads to chronic elevations inphosphorylated tau, and a shift in its disposition toward moreinsoluble, and potentially pathogenic, forms.

Example 8

Phosphatase Involvement in Restraint-Induced Tau Phosphorylation

In addition to changes in tau kinases, alterations in tau phosphataseactivity has been implicated as contributing to stress-induced tau-P(Planel et al., 2001, 2004, 2007). To determine whether similar changesmay be associated with acute or repeated restraint stress, the sameextracts used in the preceding analysis (FIG. 7A-B), were studied foralterations in the catalytic subunit of the dominant tau phosphatase,PP2A (PP2A-c). No evidence of PP2A-c within detergent-soluble RIPAfractions under was found under any experimental condition. In solubleRAB fractions, relative levels of PP2A-c from hippocampi of acutestressed mice did not differ reliably from those of controls (lanes1-3), but were significantly elevated at both 20 min (P<0.001) and 24 hrafter repeated restraint (P<0.01; lanes 4-5; see FIG. 7C). Thesefindings identify PP2A as a potential contributor to alterations intau-P, at least under repeated stress conditions.

Example 9 Stressor Specificity of CRFR Involvement

The studies relating CRFR modulation of tau-P in the hippocampus underacute stress conditions were carried out using a prototypic “emotional”stressor, physical restraint. Established animal models of anxiety(elevated plus maze exposure), fear (open field exposure, conditionedfear) and social stress (social defeat, isolation), share in common withrestraint such key features as a capacity to engage a stereotyped set ofinterconnected cell groups in the limbic forebrain, includinghippocampus (Duncan et al., 1996; Campeau et al., 1997; Martinez et al.,2002). “Emotional” is one of two major categories of stressors, and isdistinguished from the other, “physiological” type by the nature of thesensory input that registers the challenge, the extent to which theyinvoke affective responses, and the global pattern of activationalresponses they induce within the brain (Sawchenko et al., 1996; Hermanand Cullinan, 1997; Sawchenko et al., 2000; Dayas et al., 2001). Todetermine whether differential CRFR dependence extends to categoricallydistinct stressors, an immune challenge induced by bacteriallipopolysaccharide (LPS), which serves as an animal model of systemicinfection, was applied to subject mice and was examined for its effecton tauopathy in subject mice. The LPS induced stress is awell-characterized physiological stress (Turnbull et al., 1999).

Initial time course studies using the LPS induced challenge has shownrobust increases in hippocampal tau-P, peaking at 60-90 minpost-injection (FIG. 9A). Assessments of the genotype dependence of thiseffect yielded results quite distinct from those obtained with in therestraint model (FIG. 9B). Thus, acute LPS treatment resulted inenhanced phosphorylation at the PHF-1 site in wt, single and double CRFRknockouts with only subtle variation, which included a tendency fortau-P responses to be enhanced in CRFR1-deficient mice. Phosphorylationresponses at the AT8 epitope (assessed in the same extracts) were quitevariable in CRFR2 and double knockout mice, with two of three animals ineach group displaying tau-P levels at or near control values. While thebasis for this variability is uncertain, these results indicate thatneither the strong and consistent CRFR1 dependence of restraint-inducedtau-P that we have describe, nor the tendency of CRFR2 deficiency toexaggerated these responses, generalize to the LPS model. This raisesthe intriguing possibility that CRFR involvement may be limited toemotional stressors.

Example 10 Phenotypic Characterization Using a CRF-R1 ExpressingTransgenic Mouse Line

An initial step in unraveling the circuitry that provides for modulationof stress-induced tau-P by CRF signaling pathways will requireidentifying CRFR-expressing hippocampal neurons that manifeststress-induced tau-P. To facilitate this analysis, the inventors havegenerated and validated a transgenic mouse line that reports expressionof CRFR1. The enhanced green fluorescent protein (eGFP) reporterconstruct is based on a bacterial artificial chromosome (BAC) thatcontains large amounts of sequence surrounding CRFR1 such thatexpression of eGFP is controlled by promoter and enhancer sequences thatregulate endogenous receptor expression. Reporter expression in thismouse line marks the CRFR1 phenotype with high sensitivity, accuracy andcellular resolution, as determined by direct comparisons with CRFR1 mRNAexpression (FIG. 10). As with other transgenic lines generated usingthis approach, normal function of the receptor is not affected (Heintz,2001).

CRFR1-eGFP mice were exposed to acute or repeated restraint and wasexamined for dual localization of eGFP and PHF-1 immunoreactivity.Confocal microscopic analysis of doubly stained material reveals nearcomplete overlap in cellular expression of stress-induced PHF-1 withCRFR1-driven eGFP in the hilar region of the dentate gyrus, andsubstantial colocalization in pyramidal neurons of Ammon's horn. It isof interest that labeling in the transgenic extends to dendritic andaxonal process of CRFR1-expressing neurons, with prominent terminal-likelabeling seen in some cases (FIG. 10). While it cannot be assumed thatthe latter represent sites of presynaptic receptor expression, it isconsistent with this possibility. Overall, the CRFR1-eGFP mouse willprovide a sensitive and high-resolution tool of characterizing sites ofCRFR1 expression, which partially overcomes the lack of specific CRFRantisera.

Example 11 Characterization of Insoluble Tau Aggregates

NFTs consisting of PHFs are a diagnostic feature of AD. PHFs can beimaged by transmission electron microscopy of heavy metal-stainedultrathin sections or extracts of the AD brain, where they comprise˜10-20 nm diameter cylindrical filaments with helical period of −80 nm(Greenberg and Davies, 1990). They can be decorated by immunogoldlabeling to localize antibodies, such as PHF-1, to affirm the presenceof phosphorylated tau species. The inventors have validated proceduresfor labeling and imaging PHFs in extracts from AD brains (FIG. 11).

In addition, similar methods were used to image RIPA extracts ofhippocampi from mice subjected to the 14 d repeated restraint stressparadigm described above. The RIPA extracts contain the bulk ofdispersed PHFs in the AD brain (Iqbal et al., 1984; Rubenstein et al.,1986). In this material, nothing resembling PHF-like filaments wasdetected, as it was expected to. However, the samples were observedcontaining numerous structures of consistent form, rounded globularaggregates 40-100 nm in diameter (mean=69 nm), that label positively andspecifically for phosphorylated tau (PHF-1). The size and shape of thesestructures are highly reminiscent of those of aggregates assembled fromN-terminally truncated human tau under cell-free conditions (King etal., 2000). This may suggest that phosphorylated tau speciesaccumulating in the preparations are truncated or cleaved as a result ofstress, though prior Western analyses on the samples provided littleevidence of this. While tau truncated in this way cannot form filamentsin cell-free systems, it did so in vivo, and has been implicated in NFTformation and neurodegeneration (Mailliot et al., 1998; Rohn et al.,2002). Although requiring confirmation and extension, these findings mayrepresent the first evidence of pre-pathologic tau aggregates, in vivo.That the tau aggregates can occur after a limited duration ofintermittent exposure to a moderate stressor is striking, and mayindicate that stress alone is capable of initiating pathogenic changesin tau.

Example 12 Repeated Stress Induced Tau-Phosphorylation Effects areCRFR-Dependent

The CRFR status dependency of accumulation and altered solubility ofphosphorylated tau species was examined in repeatedly stressed mice.This experiment is an extension of the repeated stress study describedabove in wt, CRFR1−/−, CRFR2−/ and double knockout mice. The experimentyielded a 2×3×4×2 design with two stress conditions (acute, repeated),three post-stress time points for each (0, 20 min, 24 hr), fourgenotypes and two extraction conditions (soluble, insoluble) for a totalof 48 groups. Data for one key tau epitope (PHF-1) are shown in FIG.12A. With respect to genotype, results in wt mice fully support theanalysis above, a cumulative effect of repeated stress on tau-P at thePHF-1 site (compare acute vs repeated 24 hr lanes). In addition, theresults obtained to date extend the inventors' basic findings regardingCRFR-dependence to the repeated stress condition. Thus, under nocondition did the response of CRFR1-deficient mice exceed that ofunstressed wt controls, while responses of CRFR2 mutants were at leastcomparable, and tended to exceed, those of genetically intact animals.In addition, mice deficient in both genes fail to respond to acute orrepeated stress in a manner similar to CRFR1 mutants. With respect tothe solubility of PHF-1-phosphorylated tau under repeated stressconditions, the data shown in FIG. 12B indicate that chronic effects ofstress (i.e., 24 hr after the final exposure) are seen in both thesoluble and insoluble fractions of wt mice, that these effects tend tobe exaggerated in R2 mutants, and that stress-induced tau-P at the PHF-1site is completely lacking in CRFR1 and double knockout mice.

Collectively, these findings suggest that the genotype-dependenceobserved in acutely restrained mice extends to repeated stress effectson tau-P and solubility. Data from double knockout animals wouldindicate that CRFR2-mediated effects on these parameters areCRFR1-dependent, and likely lie upstream of them in the associatedcircuitry. The inventors will further exploit the material generated inthis study to provide a more complete characterization of repeatedstress effects on tau-P and solubility, and extend the analysis toamyloid processing and dynamics.

Example 13 Effects of CRF-R1 Antagonist on the Development of AmyloidPathology in a Mouse Model of AD

Insoluble plaques consisting of Aβ, which is derived from sequentialproteolytic processing of APP, is the second defining hallmark of ADneuropathology. Aβ is generated by sequential proteolysis of APP by βsecretase and presenilin-dependent γ secretase. Two lines of evidencewarranted extension of this study to include analyses of APP processingin repeated stress paradigms. First, while both isoforms of GSK-3 canphosphorylate tau, GSK-3β is heavily studied in tau pathology because ofits localization as a component of NFTs and its propensity to inducetangle pathology (Rankin et al., 2007). On the other hand, GSK-3α hasbeen found to be required for maximal APP processing and Aβ production(Phiel et al., 2003). The analysis of kinase modulation indicated thatGSK-3α is as potently activated by acute restraint as GSK-3β, and thatthe response of both isoforms shows a similar and differentialdependence on CRFR status. Second, a recent report documented thatrestraint stress, or central CRF administration, can increaseinterstitial fluid levels of Aβ□ albeit in APP transgenic mice (Kang etal., 2007).

Accordingly, alterations in amyloid processing affected by emotionalstress were studied. In RIPA extracts (Aβ was not detected in formicacid fractions), no significant change in Aβ42 levels was observed. TheAβ42 levels were assessed by sandwich ELISA, at 20 min after eitheracute or repeated stress exposure. However, levels of Aβ42 were observeddecreasing significantly 24 hr after the last of the repeated stressexposures (P=0.02; FIG. 13). Western analyses revealed a concomitantincrease in levels of neprilysin (an Aβ-degrading enzyme), relative tounstressed controls (P<0.01). APP levels (assessed using antibody 22C11)did not vary reliably across experimental conditions (data not shown).This pattern of results is suggestive of increased amyloid clearanceunder repeated stress conditions, and, while subtle, do provide aninitial indication that stress can impact Aβ and related enzymes inhippocampus.

A transgenic mouse model of AD was picked and used to directly assessthe role of stress and perturbations in CRF signaling in ADpathogenesis. There are a number of such mouse lines, which involvetransgenic expression of mutant forms of human amyloid precursor protein(APP), presenilin and/or tau, singly or in combination (reviewed in(Spires and Hyman, 2005; Gotz et al., 2007)). We chose one thatoverexpresses mutant human APP and presenilin-1 (PS/APP model; Jankowskyet al., 2001), which show predominantly amyloid pathology in cortex andhippocampus beginning at 3-4 months of age, with 6 months considered thetime of onset of pathology (Jankowsy et al., 2004). Alterations in tauare subtler, slower to develop, and occur in association with amyloidplaques. NFTs have not been reported in this line. Only in transgenicmodels expressing mutant tau species has tangle formation been found tooccur (Spires and Hyman, 2005; Gotz et al., 2007). The PS/APP line isideally suited to the presented study as it presents an AD-like amyloidphenotype in hippocampus and cortex, while allowing the effects ofstress experience on tau to be isolated.

Separate groups of PS/APP mice were or were not subjected to two 14-dayexposures to a chronic variable stress paradigm (CVS; see Example 1) atbeginning of their fourth and fifth months of life in the presence orabsence of concurrent treatment with the CRFR1 antagonist, antalarmin(40 mg/kg/d; ip) or vehicle. The choice of CVS as a stress regimen wasbased on the considerations that it was quite potent, resistant tohabituation, more naturalistic (Marin et al., 2007) and it afforded theopportunity for pharmacologic intervention. All animals were sacrificedat the end of the fifth month of life, shortly after the time at whichamyloid plaques begin to appear in this line.

Immunohistochemical localization of phosphorylated tau (PHF-1) revealedonly subtle variations in staining as a function of treatment status.There was no evidence of tau pathology (tangle formation), nor was itexpected to develop in animals at this early stage of diseaseprogression. Examination of plaque formation by immunohistochemical(using antibody clone 6E10; specific to the N-terminus of human Aβ) orhistochemical (thioflavin S) methods did reveal a marked effect of thesemanipulations (FIG. 14). While CVS exposure exerted no major effect onthe density of plaques in cortex and hippocampus, antalarmin treatmentsignificantly reduced in this measure in both stressed (2.49/mm²) andunstressed (1.15/mm²) groups, relative to unstressed vehicle-treatedcontrols (9.38/mm²). Thus, exposure for a limited time to the CRFR1antagonist blocked or delayed the development of amyloid pathology inthis mouse AD model. While it remains to be determined whether thiseffect will maintain in older animals, and whether drug and/or stressexposure will affect tau pathology, the results provide an additionalendorsement for considering CRFR1 as a therapeutic target in AD.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A method for delaying the onset or progression of a tauopathy, orblocking and/or delaying the onset of progression of amyloid betaamyloidosis (Aβ amyloidosis), in a subject comprising administering tothe subject an effective amount of a CRF-R1 selective antagonist and/orCRF-R2 selective agonist.
 2. The method of claim 1, wherein the CRF-R1selective antagonist has between about 10 and about 100, 1000, or 10,000fold more antagonist activity of CRF-R1 than CRF-R2.
 3. The method ofclaim 1, wherein the CRF-R1 selective antagonist has essentially noCRF-R2 antagonist activity.
 4. The method of claim 1, comprisingadministering both a CRF-R1 selective antagonist and a CRF-R2 selectiveagonist.
 5. The method of claim 4, wherein the CRF-R2 selective agonistand the CRF-R1 selective antagonist are administered separately and/orin a single formulation. 6-9. (canceled)
 10. The method of claim 1,wherein the CRF-R1 selective antagonist further comprises a centralnervous system (CNS) targeting agent. 11-15. (canceled)
 16. The methodof claim 1, wherein the tauopathy is Alzheimer's disease (AD),Amyotrophic lateral sclerosis/parkinsonism-dementia complex,Argyrophilic grain dementia, Corticobasal degeneration,Creutzfeldt-Jakob disease, Dementia pugilistica, Diffuse neurofibrillarytangles with calcificationa, Down's syndrome, Frontotemporal dementiawith parkinsonism (linked to chromosome 17),Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease,Myotonic dystrophy, Niemann-Pick disease (type C), Non-Guamanian motorneuron disease with neurofibrillary tangles, Pick's disease,Postencephalitic parkinsonism, Prion protein cerebral amyloidangiopathy, Progressive subcortical gliosis, Progressive supranuclearpalsy, Subacute sclerosing panencephalitis or Tangle only dementia.17-18. (canceled)
 19. The method of claim 1, wherein the tauopathy isfurther defined as a non-Alzheimer tauopathy.
 20. The method of claim 1,further defined as a method for delaying the onset of a tauopathy in asubject.
 21. The method of claim 20, wherein the subject is at risk fordeveloping a tauopathy.
 22. The method of claim 21, wherein the subjectcomprises a gene mutation associated with a tauopathy or comprises afamily history of tauopathy.
 23. The method of claim 21, wherein thesubject has reduced cognitive or memory function.
 24. The method ofclaim 1, wherein the subject has been diagnosed with a tauopathy. 25.The method of claim 24, wherein the subject has been diagnosed with AD.26. The method of claim 25, wherein the subject is human.
 27. (canceled)28. The method of claim 1, wherein the CRF-R1 selective antagonist isadministered topically, intravenously, intradermally, intraarterially,intraperitoneally, intracranially, intrathecally,intracerebroventricularly, mucosally, intraocularally, subcutaneously,or orally.
 29. (canceled)
 30. The method of claim 1, wherein the CRF-R1selective antagonist is administered directly to the CNS.
 31. (canceled)32. The method of claim 1, comprising administering to the subject aneffective amount of a CRF-R2 selective agonist. 33-47. (canceled) 48.The method of claim 32, wherein the CRF-R2 selective agonist isadministered topically, intravenously, intradermally, intraarterially,intraperitoneally, intracranially, intrathecally,intracerebroventricularly, mucosally, intraocularally, subcutaneously,or orally. 49-51. (canceled)
 52. A method for identifying an agent fortreating, preventing the onset or preventing progression of a tauopathycomprising: a) administering a candidate agent to an animal; b)subjecting the animal to a stress; d) determining tau phosphorylation orinsoluble tau accumulation in the CNS following stress wherein adecreased in tau phosphorylation [PP] or decreased insoluble tauaccumulation in animals treated with a candidate agent relative tocontrol animals is indicative of activity in treating, preventing theonset or preventing progression of a tauopathy. 53-77. (canceled)